Proteopedia - User contributions [en]

Sandbox Reserved 1091

2020-01-17T14:43:03Z

Lukas Jan Wyrwal:

{{Sandbox_ESBS_2019}}

== '''The serine protease from ''Aeromonas sobria'' : ASP ''' ==
<StructureSection load='3hjr' size='340' side='right' caption='General structure of ASP protein (with Ca2+ Binding Site and Disulfide Bridges)' scene=''>

== Generalities ==

The ''Aeromonas Sobria'' Serine Protease (ASP) protein is a '''serine protease''' that will cut peptide bonds after specific amino acids of a target protein. It preferentially cleaves peptide bonds that follow dibasic amino-acid residues. The kexin-like serine protease belongs to the subtilisin family ([http://en.m.wikipedia.org/wiki/Subtilase Subtilase]). The structure of ASP is similar to that of Kex2 ([[1r64]]), a protease of the subtilisin family from ''Saccharomyces cerevisiae''. <ref>PMID:2646633</ref>

This belonging to the '''subtilisin serine proteases family''' is hypothetical. Furthermore the predicted amino acid sequence reinforces this speculation. However, the size of the ASP (MW 65 kDa) is unlike other subtilisin proteases (MW 30 kDa). Also, the amino acid residues composition is different from the family’s characteristics because ASP shows unique cysteine residues that other family members don't show. Therefore we can state that it is likely that ASP belongs to the subtilisin serine proteases family, however it remains unclear. Moreover, ASP is the only prokaryotic member of the kexin-subtilisin family that is composed almost exclusively of eukaryotic proteases like Furin.<ref>Siezen RJ & Leunissen JAM (1997) Subtilase: the superfamily of subtilisin-like serine proteases. Protein Sci 6: 501–523.</ref>. ASP was shown not to be a metalloprotease because its activity is not affected by metal chelators ([http://en.wikipedia.org/wiki/Ethylenediaminetetraacetic_acid EDTA], [http://en.wikipedia.org/wiki/EGTA_(chemical) EGTA], [http://en.wikipedia.org/wiki/Phenanthroline o-phenanthroline]) or metalloprotease inhibitors ([http://en.wikipedia.org/wiki/Phosphoramidon phosphoramidon]). <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Takahisa Imamura et al. (2017)</ref>

This protein is secreted by the '''anaerobic bacterium''' [http://en.m.wikipedia.org/wiki/Aeromonas Aeromonas Sobria], which can cause potentially lethal [http://en.m.wikipedia.org/wiki/Septic_shock septic shock]. It is a clinical syndrome of potentially fatal organ dysfunction caused by a disorder in the response to infection. In septic shock, there is a critical reduction in tissue perfusion; acute multi visceral failure, including the lungs, kidneys and liver, can be observed. <ref>http://www.msdmanuals.com/professional/critical-care-medicine/sepsis-and-septic-shock/sepsis-and-septic-shock</ref>

ASP is a '''[http://en.wikipedia.org/wiki/Sepsis sepsis]-related factor'''. It can cause several dysfunctions like by inducing vascular leakage, reducing blood pressure via the activation of the [http://en.wikipedia.org/wiki/Kinin-kallikreinsystem kinin system] or promoting human plasma coagulation through the activation of [http://fr.m.wikipedia.org/wiki/Prothrombine prothrombin]. Finally, it can cause the formation of pus and edema through the action of anaphylatoxin C5a ([[4p3a]]). Gastroenteritis, and in extreme cases deuteropathy, are the main syndrome caused by infection with ''A.sobria''.

== Maturation ==

The '''precursor of ASP''' is composed of 624 amino acids. It contains a signal peptide of 24 amino acids, a catalytic domain, similar to that of subtilisin, and a P domain.
The '''maturation of ASP''' is achieved by [http://figshare.com/articles/Close-up_view_of_the_interaction_site_of_ASP_S336A_with_ORF2_/5496337/1 ORF2]. This protein plays the role of an external chaperone and is necessary for the construction of the stable ASP. Indeed, ASP doesn’t contain any propeptide that is involved in the proper folding of the protein. This is a major difference with an other protein, close to ASP : Kex2 ([[1r64]]) <ref>Structural Basis for Action of the External Chaperone for a Propeptide-deficient Serine Protease from Aeromonas sobria. Kobayashi H et al. Biol. Chem. 290(17):11130-43 (2015)</ref>

The '''ORF2 protein''' is composed of 152 amino-acids coded by the orf2 gene of 456 base pairs. The N-terminal extension and the C-terminal tail of the protein are implicated in the maturation of ASP. In fact, a complex ASP-ORF2 is formed. <ref>PMID:17951986</ref> This association requires a specific organization of ASP in the space. The <scene name='82/829344/The_p-domain/2'>P-domain</scene> of ASP doesn’t bind to ORF2 but the sixth residue from the C-terminus domain of ORF2 interacts with the non-mature ASP. In the complex, the active site of ASP is blocked. This protects the protein from degradation by others.
When the complex is formed, it moves to the extracellular space and then it dissociates. The active ASP can dissociate ORF2 and exercise its virulence activity in the cell. <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Imamura T, Murakami Y, Nitta H. Biol. Chem. 398 1055-1068 (2017)</ref>

== Secondary structure ==

The '''structure of ASP''' is very similar to that of Kex2 ([[1r64]]), but it has a unique extra-occluding region close to its active site within the subtilisin domains. This unique extra-occluding region could serve as an useful target site to facilitate the development of new antisepsis drugs.

The '''domain structure of ASP''' consists of the propeptide, the catalytic subtilisin-like domain, and the P-domain. The ASP molecule have two mean regions: an N-terminal region extending from Gly-3 to Pro-431 and forming the <scene name='82/829344/The_subtilisin_domain/2'>subtilisin domain</scene>, and a C-terminal region extending from Leu-432 to His-595 and forming the <scene name='82/829344/The_p-domain/2'>P-domain</scene>.

Moreover, we can find three <scene name='82/829344/Calcium_binding_sites/2'>Ca2+ Binding Sites</scene> in the ASP Structure (Ca1, Ca2 and Ca3). <scene name='82/829344/Ca1_et_ca2/4'>Ca1 and Ca2</scene> are situated in the N-terminal domain, while <scene name='82/829344/Ca3/3'>Ca3</scene> is situated in the C-terminal domain. These findings were assigned to ASP based on electron density, counter charges, and coordination. But contrary to Kex2 ([[1r64]]), ASP doesn't contain any Ca2+ binding sites near its catalytic site.

A schematic representation of the domains of the protein can be observed : [http://www.degruyter.com/viewimg/j/bchm.2017.398.issue-10/hsz-2016-0344/hsz-2016-0344.xml?img=graphic/j_hsz-2016-0344_fig_001.jpg '''secondary structure of ASP''']. We can see that Kex2 has the propeptide (in yellow) that is absent in ASP. The occluding subdomains in the C-terminal region of ASP are shown in dark blue.

== Domains ==

'''The Subtilisin Domain:''' It contains ten helices (alpha 1 to 10) and twelve chains (beta 1 to 10 and béta 13 to 14). The N-terminal domain of ASP seems to be like the catalytic domain of Kex2 ([[1r64]]), which is similar to those of subtilisin and other subtilisin-related proteases. This ASP catalytic site contains <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> Asp78, His115, and Ser336 residues characteristic of subtilisins. In addition, four loops (L) protrude from the N-terminal subtilisin domain of ASP : Gly3– Pro26 (<scene name='82/829344/L1/2'>L1</scene>), Asn221–Phe241 (<scene name='82/829344/L2/2'>L2</scene>), Gly300–Cys326 (<scene name='82/829344/L3/2'>L3</scene>), and Gln-377–Glu-397 (<scene name='82/829344/L4/2'>L4</scene>). L1, L2, and L3 have random coil structure, whereas L4 forms a hairpin that protrudes toward the P-domain. Moreover, two <scene name='82/829344/Disulfide_bridges/2'>disulfide bridges</scene> are formed between Cys4 and Cys24 in L1 and between Cys301 and Cys326 in L3, which stabilize those loops.

'''The P-domain:''' The core of the P-domain in ASP contains eight beta-strands (beta 16 18 23 and 26). The <scene name='82/829344/Extra_occluding_region/2'>extra occluding-region</scene> is comprised of two parts, <scene name='82/829344/Pl1/4'>pL1</scene> (Gly521–Thr525, beta 5, 6, and 12) and <scene name='82/829344/Pl2/4'>pL2</scene> (Gly557–Asn578, béta 25), and it is situated close to <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> Asp78,His115,and Ser336.

All these domains are represented schematically in the article <ref>Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor. H Kobayashi et al. J Biol Chem. 284(40): 27655–27663 (2009)</ref> : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785694/figure/F2/ '''representation 2D of ASP''']. On these figures, we can see the different domains of the protein in A and also a superposition with the Kex2. We clearly see the resemblance between both serine protease, and the extra occluding region in the C-terminal region of ASP.

== Active site ==

'''The catalytic triad:''' The [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] of ASP is composed of '''Asp78''', '''His115''' and '''Ser336'''. These amino acids are the base is the active site of the protein, where the mode of action of the serine protease takes place. A peptide can be inserted in the space of the active site. There, the amino acids of <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> will interact together and the mechanism will lead to a cut in the polypeptide.

This triad can be observed in a 2D representation of the protein : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785694/figure/F3/ '''catalytic triad of ASP''']

'''Mechanism:''' The mechanism of the reaction is the following : The histidine will react with the serine and deprotonate it. The deprotonated hydroxyl group of the serine will act as a nucleophilic species and attack the carbon from the carbonyl function on the peptide. This will lead to the formation of a tetrahedral intermediate. The regeneration of the carbonyl group will be followed by the release of one part of the peptide, with an amine group at its extremity. Then, a second tetrahedral intermediate will be formed, but with the attack of a deprotonated water molecule. In the end, the regeneration of the active site will be done with the release of the part of the peptide with a carboxyl extremity. '''The polypeptide is also cut in two parts and the target protein isn't functional anymore'''. <ref>http://fr.wikipedia.org/wiki/Fichier:Serine_protease_mechanism_by_snellios.png</ref>

A schematic representation of the mechanism with the involved amino acids can be found under the following link : [http://fr.wikipedia.org/wiki/Fichier:Serine_protease_mechanism_by_snellios.png '''mechanism of the reaction''' ]

== Properties ==

ASP has its highest activity at pH 7,5 and loses it after heating at 60° for 10 minutes. <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Imamura T, Murakami Y, Nitta H. Biol. Chem. 398 1055-1068 (2017)</ref>

The '''peptide bonds''' were shown to be cleaved when two basic residues were in sequence. A Lys residue at positions P1 and P2 relative to the cleavage site is prefered. If an Arg residue is at P4 position the substrate cleavage will be enhanced. <ref>Cleavage specificity of serine protease of Aeromonas sobria, a member of the kexin family of subtilases., H. Kobayashi, Okayama University, Japan,FEMS Microbiology Letters, Volume 256, Issue 1, March 2006, Pages 165–170,</ref>

Experiments have been done in order to establish the '''sensitivity of ASP to proteases'''. In has been found that the ASP protease activity was strongly attenuated by serine protease inhibitors ([http://en.wikipedia.org/wiki/Diisopropyl_fluorophosphate DFP], [http://fr.wikipedia.org/wiki/Fluorure_de_4-(2-aminoéthyl)benzènesulfonyle AEBSF]). Moreover, a soybean [http://en.wikipedia.org/wiki/Trypsin_inhibitor trypsin inhibitor] was shown not to block the proteolytic action of ASP itself but could inhibit the [http://en.m.wikipedia.org/wiki/Vascular_permeability vascular permeability] enhancing activity that follows after injection of ASP into epithelial cells. <ref>Physicochemical and biological properties od an extracellular serine protease od Aeromonas sobria. Ritsuko Yokoyama, Yoshio Fujii et al. Japan (2002)</ref>

The experimental finding suggests that epithelial trypsin-like proteases mediate the reaction causing enhanced vascular permeability. It is likely that ASP stimulates the secretion and maturation of epithelial trypsin proteases, thus enhancing vascular permeability. ASP could stimulate the [http://en.wikipedia.org/wiki/Bradykinin bradykinin]-releasing pathway, thus stimulating mast cells to release histamine and further enhance the vascular permeability.
'''Antihistaminic agents''' ([http://en.wikipedia.org/wiki/Diphenhydramine diphenhydramine] and [http://en.wikipedia.org/wiki/Mepyramine pyrilamine]) were shown to efficiently inhibit vascular permeability enhancing the activity of the ASP. It is very likely that the vascular permeability enhancement is related to the release of [http://en.wikipedia.org/wiki/Histamine histamine] from [http://en.wikipedia.org/wiki/Mast_cell mast cells].
Through histopathological examinations it was shown that mast cells appeared around the injection site, confirming the role of histamine as a key factor.<ref>Physicochemical and biological properties od an extracellular serine protease od Aeromonas sobria. Ritsuko Yokoyama, Yoshio Fujii et al. Japan (2002)</ref>

== Impact on human body ==

The '''predominant infection vector''' is through exposure to water harbouring ''Aeromonas sobria''. <ref>Joseph, S. W., O. P. Daily, W. S. Hunt, R. J. Seidler, D. A. Allen, and R. R. Colwell. 1979. Aeromonas primary wound '''infection of a diver in polluted waters'''. J. Clin. Microbiol. 10:46-49.</ref>
The most common form of disease is one where ''A.sobria'' pathogen adheres to the surface of the intestine causing painful diarrhea, also known as [http://en.wikipedia.org/wiki/Gastroenteritis gastroenteritis]. The enterotoxin activity of the [http://en.wikipedia.org/wiki/Hemolysin hemolysin] [http://en.wikipedia.org/wiki/Virulence_factor virulence factors] of ''A.sobria'' contributes to those symptoms. However, the mortality due to intestinal disease type of infection is low compared to the non-intestinal diseases caused by the ''A.sobria'' infection.
Pili facilitate the adherence of ''Aeromonas'' to human buccal cells. <ref>Cleavage specificity of serine protease of Aeromonas sobria, a member of the kexin family of subtilases., H. Kobayashi, Okayama University, Japan,FEMS Microbiology Letters, Volume 256, Issue 1, March 2006, Pages 165–170,</ref>
Once invaded the intestine epithelial cells, Aeromonas can reach any organ via the blood. Multiple virulence factor than promote their pathogenicity.

The nonintestinal form of the disease reports symptoms such as [http://en.wikipedia.org/wiki/Sepsis septicemia], lesions of skin and soft tissues as well as [http://en.wikipedia.org/wiki/Meningitis meningitis], often ending fatally. That leads to a crucial reduction in tissue perfusion followed by fatal organ disfunction.

The ASP induced '''proteolysis digestion of proteins''' like [http://en.wikipedia.org/wiki/Kininogen kininogen], [http://en.wikipedia.org/wiki/Thrombin prothrombin], [http://en.wikipedia.org/wiki/Fibrinogen fibrinogen] or [http://en.wikipedia.org/wiki/Prekallikrein prekallikrein] at restricted sites generates fragments, expressing their own activity and therefore inducing specific physiological reactions. The [http://en.wikipedia.org/wiki/Kinin–kallikrein_system kinin system] activation, for example, reduces the blood pressure while the prothrombin system promotes plasma coagulation.

In the '''kallikrein/kinin system''', the ASP induce the production of kinin from kininogen (low MW or high MW). The kinin also release caused edema at the infection site and shock in the circulation. The schematic representation of this action is represented : [http://www.degruyter.com/viewimg/j/bchm.2017.398.issue-10/hsz-2016-0344/hsz-2016-0344.xml?img=graphic/j_hsz-2016-0344_fig_002.jpg '''acting points of ASP in the kinin system''' ]

Experiments have been done in order to try to reduce the virulence activity of ASP. It has been demonstrated that the [http://fr.wikipedia.org/wiki/Alpha-2_macroglobuline α2-macroglobulin], a plasma protein, can limit ASP activity. This protein can bind to ASP which is also inactivated. <ref>Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin. Murakami Y et al. Biol Chem. 393(10):1193-200 (2012)</ref>

</StructureSection>

== References ==
<references/>
<scene name='82/829344/The_p-domain/1'>Text To Be Displayed</scene>

Sandbox Reserved 1091

2020-01-17T14:37:12Z

Lukas Jan Wyrwal:

{{Sandbox_ESBS_2019}}

== '''The serine protease from ''Aeromonas sobria'' : ASP ''' ==
<StructureSection load='3hjr' size='340' side='right' caption='General structure of ASP protein (with Ca2+ Binding Site and Disulfide Bridges)' scene=''>

== Generalities ==

The ''Aeromonas Sobria'' Serine Protease (ASP) protein is a '''serine protease''' that will cut peptide bonds after specific amino acids of a target protein. It preferentially cleaves peptide bonds that follow dibasic amino-acid residues. The kexin-like serine protease belongs to the subtilisin family ([http://en.m.wikipedia.org/wiki/Subtilase Subtilase]). The structure of ASP is similar to that of Kex2 ([[1r64]]), a protease of the subtilisin family from ''Saccharomyces cerevisiae''. <ref>PMID:2646633</ref>

This belonging to the '''subtilisin serine proteases family''' is hypothetical. Furthermore the predicted amino acid sequence reinforces this speculation. However, the size of the ASP (MW 65 kDa) is unlike other subtilisin proteases (MW 30 kDa). Also, the amino acid residues composition is different from the family’s characteristics because ASP shows unique cysteine residues that other family members don't show. Therefore we can state that it is likely that ASP belongs to the subtilisin serine proteases family, however it remains unclear. Moreover, ASP is the only prokaryotic member of the kexin-subtilisin family that is composed almost exclusively of eukaryotic proteases like Furin.<ref>Siezen RJ & Leunissen JAM (1997) Subtilase: the superfamily of subtilisin-like serine proteases. Protein Sci 6: 501–523.</ref>. ASP was shown not to be a metalloprotease because its activity is not affected by metal chelators ([http://en.wikipedia.org/wiki/Ethylenediaminetetraacetic_acid EDTA], [http://en.wikipedia.org/wiki/EGTA_(chemical) EGTA], [http://en.wikipedia.org/wiki/Phenanthroline o-phenanthroline]) or metalloprotease inhibitors ([http://en.wikipedia.org/wiki/Phosphoramidon phosphoramidon]). <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Takahisa Imamura et al. (2017)</ref>

This protein is secreted by the '''anaerobic bacterium''' [http://en.m.wikipedia.org/wiki/Aeromonas Aeromonas Sobria], which can cause potentially lethal [http://en.m.wikipedia.org/wiki/Septic_shock septic shock]. It is a clinical syndrome of potentially fatal organ dysfunction caused by a disorder in the response to infection. In septic shock, there is a critical reduction in tissue perfusion; acute multi visceral failure, including the lungs, kidneys and liver, can be observed. <ref>http://www.msdmanuals.com/professional/critical-care-medicine/sepsis-and-septic-shock/sepsis-and-septic-shock</ref>

ASP is a '''[http://en.wikipedia.org/wiki/Sepsis sepsis]-related factor'''. It can cause several dysfunctions like by inducing vascular leakage, reducing blood pressure via the activation of the [http://en.wikipedia.org/wiki/Kinin-kallikreinsystem kinin system] or promoting human plasma coagulation through the activation of [http://fr.m.wikipedia.org/wiki/Prothrombine prothrombin]. Finally, it can cause the formation of pus and edema through the action of anaphylatoxin C5a ([[4p3a]]). Gastroenteritis, and in extreme cases deuteropathy, are the main syndrome caused by infection with ''A.sobria''.

== Maturation ==

The '''precursor of ASP''' is composed of 624 amino acids. It contains a signal peptide of 24 amino acids, a catalytic domain, similar to that of subtilisin, and a P domain.
The '''maturation of ASP''' is achieved by [http://figshare.com/articles/Close-up_view_of_the_interaction_site_of_ASP_S336A_with_ORF2_/5496337/1 ORF2]. This protein plays the role of an external chaperone and is necessary for the construction of the stable ASP. Indeed, ASP doesn’t contain any propeptide that is involved in the proper folding of the protein. This is a major difference with an other protein, close to ASP : Kex2 ([[1r64]]) <ref>Structural Basis for Action of the External Chaperone for a Propeptide-deficient Serine Protease from Aeromonas sobria. Kobayashi H et al. Biol. Chem. 290(17):11130-43 (2015)</ref>

The '''ORF2 protein''' is composed of 152 amino-acids coded by the orf2 gene of 456 base pairs. The N-terminal extension and the C-terminal tail of the protein are implicated in the maturation of ASP. In fact, a complex ASP-ORF2 is formed. <ref>PMID:17951986</ref> This association requires a specific organization of ASP in the space. The <scene name='82/829344/The_p-domain/2'>P-domain</scene> of ASP doesn’t bind to ORF2 but the sixth residue from the C-terminus domain of ORF2 interacts with the non-mature ASP. In the complex, the active site of ASP is blocked. This protects the protein from degradation by others.
When the complex is formed, it moves to the extracellular space and then it dissociates. The active ASP can dissociate ORF2 and exercise its virulence activity in the cell. <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Imamura T, Murakami Y, Nitta H. Biol. Chem. 398 1055-1068 (2017)</ref>

== Secondary structure ==

The '''structure of ASP''' is very similar to that of Kex2 ([[1r64]]), but it has a unique extra-occluding region close to its active site within the subtilisin domains. This unique extra-occluding region could serve as an useful target site to facilitate the development of new antisepsis drugs.

The '''domain structure of ASP''' consists of the propeptide, the catalytic subtilisin-like domain, and the P-domain. The ASP molecule have two mean regions: an N-terminal region extending from Gly-3 to Pro-431 and forming the <scene name='82/829344/The_subtilisin_domain/2'>subtilisin domain</scene>, and a C-terminal region extending from Leu-432 to His-595 and forming the <scene name='82/829344/The_p-domain/2'>P-domain</scene>.

Moreover, we can find three <scene name='82/829344/Calcium_binding_sites/2'>Ca2+ Binding Sites</scene> in the ASP Structure (Ca1, Ca2 and Ca3). <scene name='82/829344/Ca1_et_ca2/4'>Ca1 and Ca2</scene> are situated in the N-terminal domain, and <scene name='82/829344/Ca3/3'>Ca3</scene> is situated in the C-terminal domain. It were assigned to ASP based on electron density, counter charges, and coordination. But in contrary to Kex2 ([[1r64]]), ASP contains no Ca2+ binding sites near its catalytic site.

A schematic representation of the domains of the protein can be observed : [http://www.degruyter.com/viewimg/j/bchm.2017.398.issue-10/hsz-2016-0344/hsz-2016-0344.xml?img=graphic/j_hsz-2016-0344_fig_001.jpg '''secondary structure of ASP''']. We can see that Kex2 has the propeptide (in yellow) that is absent in ASP. The occluding subdomains in the C-terminal region of ASP are shown in dark blue.

== Domains ==

'''The Subtilisin Domain:''' It contains ten helices (alpha 1 to 10) and twelve chains (beta 1 to 10 and béta 13 to 14). The N-terminal domain of ASP seems to be like the catalytic domain of Kex2 ([[1r64]]), which is similar to those of subtilisin and other subtilisin-related proteases. This ASP catalytic site contains <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> Asp78, His115, and Ser336 residues characteristic of subtilisins. In addition, four loops (L) protrude from the N-terminal subtilisin domain of ASP : Gly3– Pro26 (<scene name='82/829344/L1/2'>L1</scene>), Asn221–Phe241 (<scene name='82/829344/L2/2'>L2</scene>), Gly300–Cys326 (<scene name='82/829344/L3/2'>L3</scene>), and Gln-377–Glu-397 (<scene name='82/829344/L4/2'>L4</scene>). L1, L2, and L3 have random coil structure, whereas L4 forms a hairpin that protrudes toward the P-domain. Moreover, two <scene name='82/829344/Disulfide_bridges/2'>disulfide bridges</scene> are formed between Cys4 and Cys24 in L1 and between Cys301 and Cys326 in L3, which stabilize those loops.

'''The P-domain:''' The core of the P-domain in ASP contains eight beta-strands (beta 16 18 23 and 26). The <scene name='82/829344/Extra_occluding_region/2'>extra occluding-region</scene> is comprised of two parts, <scene name='82/829344/Pl1/4'>pL1</scene> (Gly521–Thr525, beta 5, 6, and 12) and <scene name='82/829344/Pl2/4'>pL2</scene> (Gly557–Asn578, béta 25), and it is situated close to <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> Asp78,His115,and Ser336.

All these domains are represented schematically in the article <ref>Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor. H Kobayashi et al. J Biol Chem. 284(40): 27655–27663 (2009)</ref> : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785694/figure/F2/ '''representation 2D of ASP''']. On these figures, we can see the different domains of the protein in A and also a superposition with the Kex2. We clearly see the resemblance between both serine protease, and the extra occluding region in the C-terminal region of ASP.

== Active site ==

'''The catalytic triad:''' The [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] of ASP is composed of '''Asp78''', '''His115''' and '''Ser336'''. These amino acids are the base is the active site of the protein, where the mode of action of the serine protease takes place. A peptide can be inserted in the space of the active site. There, the amino acids of <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> will interact together and the mechanism will lead to a cut in the polypeptide.

This triad can be observed in a 2D representation of the protein : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785694/figure/F3/ '''catalytic triad of ASP''']

'''Mechanism:''' The mechanism of the reaction is the following : The histidine will react with the serine and deprotonate it. The deprotonated hydroxyl group of the serine will act as a nucleophilic species and attack the carbon from the carbonyl function on the peptide. This will lead to the formation of a tetrahedral intermediate. The regeneration of the carbonyl group will be followed by the release of one part of the peptide, with an amine group at its extremity. Then, a second tetrahedral intermediate will be formed, but with the attack of a deprotonated water molecule. In the end, the regeneration of the active site will be done with the release of the part of the peptide with a carboxyl extremity. '''The polypeptide is also cut in two parts and the target protein isn't functional anymore'''. <ref>http://fr.wikipedia.org/wiki/Fichier:Serine_protease_mechanism_by_snellios.png</ref>

A schematic representation of the mechanism with the involved amino acids can be found under the following link : [http://fr.wikipedia.org/wiki/Fichier:Serine_protease_mechanism_by_snellios.png '''mechanism of the reaction''' ]

== Properties ==

ASP has its highest activity at pH 7,5 and loses it after heating at 60° for 10 minutes. <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Imamura T, Murakami Y, Nitta H. Biol. Chem. 398 1055-1068 (2017)</ref>

The '''peptide bonds''' were shown to be cleaved when two basic residues were in sequence. A Lys residue at positions P1 and P2 relative to the cleavage site is prefered. If an Arg residue is at P4 position the substrate cleavage will be enhanced. <ref>Cleavage specificity of serine protease of Aeromonas sobria, a member of the kexin family of subtilases., H. Kobayashi, Okayama University, Japan,FEMS Microbiology Letters, Volume 256, Issue 1, March 2006, Pages 165–170,</ref>

Experiments have been done in order to establish the '''sensitivity of ASP to proteases'''. In has been found that the ASP protease activity was strongly attenuated by serine protease inhibitors ([http://en.wikipedia.org/wiki/Diisopropyl_fluorophosphate DFP], [http://fr.wikipedia.org/wiki/Fluorure_de_4-(2-aminoéthyl)benzènesulfonyle AEBSF]). Moreover, a soybean [http://en.wikipedia.org/wiki/Trypsin_inhibitor trypsin inhibitor] was shown not to block the proteolytic action of ASP itself but could inhibit the [http://en.m.wikipedia.org/wiki/Vascular_permeability vascular permeability] enhancing activity that follows after injection of ASP into epithelial cells. <ref>Physicochemical and biological properties od an extracellular serine protease od Aeromonas sobria. Ritsuko Yokoyama, Yoshio Fujii et al. Japan (2002)</ref>

The experimental finding suggests that epithelial trypsin-like proteases mediate the reaction causing enhanced vascular permeability. It is likely that ASP stimulates the secretion and maturation of epithelial trypsin proteases, thus enhancing vascular permeability. ASP could stimulate the [http://en.wikipedia.org/wiki/Bradykinin bradykinin]-releasing pathway, thus stimulating mast cells to release histamine and further enhance the vascular permeability.
'''Antihistaminic agents''' ([http://en.wikipedia.org/wiki/Diphenhydramine diphenhydramine] and [http://en.wikipedia.org/wiki/Mepyramine pyrilamine]) were shown to efficiently inhibit vascular permeability enhancing the activity of the ASP. It is very likely that the vascular permeability enhancement is related to the release of [http://en.wikipedia.org/wiki/Histamine histamine] from [http://en.wikipedia.org/wiki/Mast_cell mast cells].
Through histopathological examinations it was shown that mast cells appeared around the injection site, confirming the role of histamine as a key factor.<ref>Physicochemical and biological properties od an extracellular serine protease od Aeromonas sobria. Ritsuko Yokoyama, Yoshio Fujii et al. Japan (2002)</ref>

== Impact on human body ==

The '''predominant infection vector''' is through exposure to water harbouring ''Aeromonas sobria''. <ref>Joseph, S. W., O. P. Daily, W. S. Hunt, R. J. Seidler, D. A. Allen, and R. R. Colwell. 1979. Aeromonas primary wound '''infection of a diver in polluted waters'''. J. Clin. Microbiol. 10:46-49.</ref>
The most common form of disease is one where ''A.sobria'' pathogen adheres to the surface of the intestine causing painful diarrhea, also known as [http://en.wikipedia.org/wiki/Gastroenteritis gastroenteritis]. The enterotoxin activity of the [http://en.wikipedia.org/wiki/Hemolysin hemolysin] [http://en.wikipedia.org/wiki/Virulence_factor virulence factors] of ''A.sobria'' contributes to those symptoms. However, the mortality due to intestinal disease type of infection is low compared to the non-intestinal diseases caused by the ''A.sobria'' infection.
Pili facilitate the adherence of ''Aeromonas'' to human buccal cells. <ref>Cleavage specificity of serine protease of Aeromonas sobria, a member of the kexin family of subtilases., H. Kobayashi, Okayama University, Japan,FEMS Microbiology Letters, Volume 256, Issue 1, March 2006, Pages 165–170,</ref>
Once invaded the intestine epithelial cells, Aeromonas can reach any organ via the blood. Multiple virulence factor than promote their pathogenicity.

The nonintestinal form of the disease reports symptoms such as [http://en.wikipedia.org/wiki/Sepsis septicemia], lesions of skin and soft tissues as well as [http://en.wikipedia.org/wiki/Meningitis meningitis], often ending fatally. That leads to a crucial reduction in tissue perfusion followed by fatal organ disfunction.

The ASP induced '''proteolysis digestion of proteins''' like [http://en.wikipedia.org/wiki/Kininogen kininogen], [http://en.wikipedia.org/wiki/Thrombin prothrombin], [http://en.wikipedia.org/wiki/Fibrinogen fibrinogen] or [http://en.wikipedia.org/wiki/Prekallikrein prekallikrein] at restricted sites generates fragments, expressing their own activity and therefore inducing specific physiological reactions. The [http://en.wikipedia.org/wiki/Kinin–kallikrein_system kinin system] activation, for example, reduces the blood pressure while the prothrombin system promotes plasma coagulation.

In the '''kallikrein/kinin system''', the ASP induce the production of kinin from kininogen (low MW or high MW). The kinin also release caused edema at the infection site and shock in the circulation. The schematic representation of this action is represented : [http://www.degruyter.com/viewimg/j/bchm.2017.398.issue-10/hsz-2016-0344/hsz-2016-0344.xml?img=graphic/j_hsz-2016-0344_fig_002.jpg '''acting points of ASP in the kinin system''' ]

Experiments have been done in order to try to reduce the virulence activity of ASP. It has been demonstrated that the [http://fr.wikipedia.org/wiki/Alpha-2_macroglobuline α2-macroglobulin], a plasma protein, can limit ASP activity. This protein can bind to ASP which is also inactivated. <ref>Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin. Murakami Y et al. Biol Chem. 393(10):1193-200 (2012)</ref>

</StructureSection>

== References ==
<references/>
<scene name='82/829344/The_p-domain/1'>Text To Be Displayed</scene>

Sandbox Reserved 1091

2020-01-16T18:42:36Z

Lukas Jan Wyrwal:

{{Sandbox_ESBS_2019}}

== '''The serine protease from ''Aeromonas sobria'' : ASP ''' ==
<StructureSection load='3hjr' size='340' side='right' caption='General structure of ASP protein (with Ca2+ Binding Site and Disulfide Bridges)' scene=''>

== Generalities ==

The ''Aeromonas Sobria Serine Protease'' ASP protein is a '''serine protease''' that will cut peptide bonds after specific amino acids of a target protein. It preferentially cleaves peptide bonds that follow dibasic amino-acid residues. The kexin-like serine protease belongs to the subtilisin family ([http://en.m.wikipedia.org/wiki/Subtilase Subtilase]). The structure of ASP is similar to that of Kex2 ([[1r64]]), a protease of the subtilisin family from Saccharomyces cerevisiae. <ref>PMID:2646633</ref>

This belonging to the subtilisin serine proteases family is hypothetical. Furthermore the predicted amino acid sequence reinforces this speculation. However, the size of the ASP (MW 65 kDa) is unlike other subtilisin proteases (MW 30 kDa). Also, the amino acid residues composition is different from the family’s characteristics because ASP shows unique cysteine residues that other family members don't show. Therefore we can state that it is likely that ASP belongs to the subtilisin serine proteases family, however it remains unclear.
Moreover, ASP is the only prokaryotic memebr of the kexin-subtilisin family that is composed almost exclusively of eukaryotic proteases like Furin.<ref>Siezen RJ & Leunissen JAM (1997) Subtilase: the superfamily of subtilisin-like serine proteases. Protein Sci 6: 501–523.</ref>
ASP was shown not to be a metalloprotease because its activity is not affected by metal chelators ([http://en.wikipedia.org/wiki/Ethylenediaminetetraacetic_acid EDTA], [http://en.wikipedia.org/wiki/EGTA_(chemical) EGTA], [http://en.wikipedia.org/wiki/Phenanthroline o-phenanthroline]) or metalloprotease inhibitors ([http://en.wikipedia.org/wiki/Phosphoramidon phosphoramidon]). <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Takahisa Imamura et al. (2017)</ref>

This protein is secreted by the Anaerobic bacterium [http://en.m.wikipedia.org/wiki/Aeromonas Aeromonas Sobria], which can cause potentially lethal [http://en.m.wikipedia.org/wiki/Septic_shock septic shock]. It is a clinical syndrome of potentially fatal organ dysfunction caused by a disorder in the response to infection. In septic shock, there is a critical reduction in tissue perfusion; acute multi visceral failure, including the lungs, kidneys and liver, can be observed. <ref>http://www.msdmanuals.com/professional/critical-care-medicine/sepsis-and-septic-shock/sepsis-and-septic-shock</ref>

ASP is a '''[http://en.wikipedia.org/wiki/Sepsis sepsis]-related factor'''. It can cause several dysfunctions like by inducing vascular leakage, reducing blood pressure via the activation of the [http://en.wikipedia.org/wiki/Kinin-kallikreinsystem kinin system] or promoting human plasma coagulation through the activation of [http://fr.m.wikipedia.org/wiki/Prothrombine prothrombin]. Finally, it can cause the formation of pus and edema through the action of anaphylatoxin C5a ([[4p3a]]). Gastroenteritis, and in extreme cases deuteropathy, are the main syndrome caused by infection with ''A.sobria''.

== Maturation ==

The precursor of ASP is composed of 624 amino acids. It contains a signal peptide of 24 amino acids, a catalytic domain, similar to that of subtilisin, and a P domain.
The '''maturation of ASP''' is achieved by ORF2. This protein plays the role of an external chaperone and is necessary for the construction of the stable ASP. Indeed, ASP doesn’t contain any propeptide that is involved in the proper folding of the protein. This is a major difference with an other protein, close to ASP : Kex2 ([[1r64]]) <ref>Structural Basis for Action of the External Chaperone for a Propeptide-deficient Serine Protease from Aeromonas sobria. Kobayashi H et al. Biol. Chem. 290(17):11130-43 (2015)</ref>

The ORF2 protein is composed of 152 amino-acids coded by the orf2 gene of 456 base pairs. The N-terminal extension and the C-terminal tail of the protein are implicated in the maturation of ASP. In fact, a complex ASP-ORF2 is formed. This association requires a specific organization of ASP in the space. The <scene name='82/829344/The_p-domain/2'>P-domain</scene> of ASP doesn’t bind to ORF2 but the sixth residue from the C-terminus domain of ORF2 interacts with the non-mature ASP. In the complex, the active site of ASP is blocked. This protects the protein from degradation by others.
When the complex is formed, it moves to the extracellular space and then it dissociates. The active ASP can dissociate ORF2 and exercise its virulence activity in the cell. <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Imamura T, Murakami Y, Nitta H. Biol. Chem. 398 1055-1068 (2017)</ref>

== Secondary structure ==

The '''structure of ASP''' is very similar to that of Kex2 ([[1r64]]), but it has a unique extra-occluding region close to its active site within the subtilisin domains. This extra-occluding region is unique and it could serve as a useful target to make the development of new antisepsis drugs easier.

The '''domain structure of ASP''' consists of the propeptide, the catalytic subtilisin-like domain, and the P-domain. The ASP molecule have two mean regions: an N-terminal region extending from Gly-3 to Pro-431 and forming the <scene name='82/829344/The_subtilisin_domain/2'>subtilisin domain</scene>, and a C-terminal region extending from Leu-432 to His-595 and forming the <scene name='82/829344/The_p-domain/2'>P-domain</scene>.

Moreover, we can find three <scene name='82/829344/Calcium_binding_sites/2'>Ca2+ Binding Sites</scene> in the ASP Structure (Ca1, Ca2 and Ca3). <scene name='82/829344/Ca1_et_ca2/4'>Ca1 and Ca2</scene> are situated in the N-terminal domain, and <scene name='82/829344/Ca3/3'>Ca3</scene> is situated in the C-terminal domain. It were assigned to ASP based on electron density, counter charges, and coordination. But in contrary to Kex2 ([[1r64]]), ASP contains no Ca2+ binding sites near its catalytic site.

A schematic representation of the domains of the protein can be observed : [http://www.degruyter.com/viewimg/j/bchm.2017.398.issue-10/hsz-2016-0344/hsz-2016-0344.xml?img=graphic/j_hsz-2016-0344_fig_001.jpg '''secondary structure of ASP''']
We can see that Kex2 has the propeptide (in yellow) that is absent in ASP. The occluding subdomains in the C-terminal region of ASP are shown in dark blue.

== Domains ==

'''The Subtilisin Domain:''' It contains ten helices (alpha 1 to 10) and twelve chains (beta 1 to 10 and béta 13 to 14). The N-terminal domain of ASP seems to be like the catalytic domain of Kex2 ([[1r64]]), which is similar to those of subtilisin and other subtilisin-related proteases. This ASP catalytic site contains <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> Asp78, His115, and Ser336 residues characteristic of subtilisins. In addition, four loops (L) protrude from the N-terminal subtilisin domain of ASP : Gly3– Pro26 (<scene name='82/829344/L1/2'>L1</scene>), Asn221–Phe241 (<scene name='82/829344/L2/2'>L2</scene>), Gly300–Cys326 (<scene name='82/829344/L3/2'>L3</scene>), and Gln-377–Glu-397 (<scene name='82/829344/L4/2'>L4</scene>). L1, L2, and L3 have random coil structure, whereas L4 forms a hairpin that protrudes toward the P-domain. Moreover, two <scene name='82/829344/Disulfide_bridges/2'>disulfide bridges</scene> are formed between Cys4 and Cys24 in L1 and between Cys301 and Cys326 in L3, which stabilize those loops.

'''The P-domain:''' The core of the P-domain in ASP contains eight beta-strands (beta 16 18 23 and 26). The <scene name='82/829344/Extra_occluding_region/2'>extra occluding-region</scene> is comprised of two parts, <scene name='82/829344/Pl1/3'>pL1</scene>(Gly521–Thr525, beta 5, 6, and 12) and <scene name='82/829344/Pl2/3'>pL2</scene> (Gly557–Asn578, béta 25), and it is situated close to <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> Asp78,His115,and Ser336.

All these domains are represented schematically in the article <ref>Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor. H Kobayashi et al. J Biol Chem. 284(40): 27655–27663 (2009)</ref> : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785694/figure/F2/ '''representation 2D of ASP''']

On these figures, we can see the different domains of the protein in A and also a superposition with the Kex2. We clearly see the resemblance between both serine protease, and the extra occluding region in the C-terminal region of ASP.

== Active site ==

'''The catalytic triad:''' The [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] of ASP is composed of '''Asp78''', '''His115''' and '''Ser336'''. These amino acids are the base is the active site of the protein, where the mode of action of the serine protease takes place. A peptide can be inserted in the space of the active site. There, the amino acids of <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> will interact together and the mechanism will lead to a cut in the polypeptide.

This triad can be observed in a 2D representation of the protein : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785694/figure/F3/ '''catalytic triad of ASP''']

'''Mechanism:''' The mechanism of the reaction is the following : The histidine will react with the serine and deprotonate it. The deprotonated hydroxyl group of the serine will act as a nucleophilic species and attack the carbon from the carbonyl function on the peptide. This will lead to the formation of a tetrahedral intermediate. The regeneration of the carbonyl group will be followed by the release of one part of the peptide, with an amine group at its extremity. Then, a second tetrahedral intermediate will be formed, but with the attack of a deprotonated water molecule. In the end, the regeneration of the active site will be done with the release of the part of the peptide with a carboxyl extremity. '''The polypeptide is also cut in two parts and the target protein isn't functional anymore'''. <ref>http://fr.wikipedia.org/wiki/Fichier:Serine_protease_mechanism_by_snellios.png</ref>

A schematic representation of the mechanism with the involved amino acids can be found under the following link : [http://fr.wikipedia.org/wiki/Fichier:Serine_protease_mechanism_by_snellios.png '''mechanism of the reaction''' ]

== Properties ==

ASP has its highest activity at pH 7,5 and loses it after heating at 60° for 10 minutes. <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Imamura T, Murakami Y, Nitta H. Biol. Chem. 398 1055-1068 (2017)</ref>

The peptide bonds were shown to be cleaved when two basic residues were in sequence. A Lys residue at positions P1 and P2 relative to the cleavage site is prefered. If an Arg residue is at P4 position the substrate cleavage will be enhanced. <ref>Cleavage specificity of serine protease of Aeromonas sobria, a member of the kexin family of subtilases., H. Kobayashi, Okayama University, Japan,FEMS Microbiology Letters, Volume 256, Issue 1, March 2006, Pages 165–170,</ref>

Experiments have been done in order to establish the sensitivity of ASP to proteases. In has been found that the ASP protease activity was strongly attenuated by serine protease inhibitors ([http://en.wikipedia.org/wiki/Diisopropyl_fluorophosphate DFP], [http://fr.wikipedia.org/wiki/Fluorure_de_4-(2-aminoéthyl)benzènesulfonyle AEBSF]). Moreover, a soybean [http://en.wikipedia.org/wiki/Trypsin_inhibitor trypsin inhibitor] was shown not to block the proteolytic action of ASP itself but could inhibit the [http://en.m.wikipedia.org/wiki/Vascular_permeability vascular permeability] enhancing activity that follows after injection of ASP into epithelial cells. <ref>Physicochemical and biological properties od an extracellular serine protease od Aeromonas sobria. Ritsuko Yokoyama, Yoshio Fujii et al. Japan (2002)</ref>

This experimental finding suggests that epithelial trypsin-like proteases mediate the reaction causing enhanced vascular permeability. It is likely that ASP stimulates the secretion and maturation of epithelial trypsin proteases, thus enhancing vascular permeability. ASP could stimulate the [http://en.wikipedia.org/wiki/Bradykinin bradykinin]-releasing pathway, thus stimulating mast cells to release histamine and further enhance the vascular permeability.
Antihistaminic agents ([http://en.wikipedia.org/wiki/Diphenhydramine diphenhydramine] and [http://en.wikipedia.org/wiki/Mepyramine pyrilamine]) were shown to efficiently inhibit vascular permeability enhancing the activity of the ASP. It is very likely that the vascular permeability enhancement is related to the release of [http://en.wikipedia.org/wiki/Histamine histamine] from [http://en.wikipedia.org/wiki/Mast_cell mast cells].
Through histopathological examinations it was shown that mast cells appeared around the injection site, confirming the role of histamine as a key factor.<ref>Physicochemical and biological properties od an extracellular serine protease od Aeromonas sobria. Ritsuko Yokoyama, Yoshio Fujii et al. Japan (2002)</ref>

== Impact on human body ==

The predominant infection vector is through exposure to water harbouring ''Aeromonas sobria''. <ref>Joseph, S. W., O. P. Daily, W. S. Hunt, R. J. Seidler, D. A. Allen, and R. R. Colwell. 1979. Aeromonas primary wound infection of a diver in polluted waters. J. Clin. Microbiol. 10:46-49.</ref>
The most common form of disease is one where ''A.sobria'' pathogen adheres to the surface of the intestine causing painful diarrhea, also known as [http://en.wikipedia.org/wiki/Gastroenteritis gastroenteritis]. The enterotoxin activity of the [http://en.wikipedia.org/wiki/Hemolysin hemolysin] [http://en.wikipedia.org/wiki/Virulence_factor virulence factors] of ''A.sobria'' contributes to those symptoms. However, the mortality due to intestinal disease type of infection is low compared to the non-intestinal diseases caused by the ''A.sobria'' infection.
Pili facilitate the adherence of ''Aeromonas'' to human buccal cells. <ref>Cleavage specificity of serine protease of Aeromonas sobria, a member of the kexin family of subtilases., H. Kobayashi, Okayama University, Japan,FEMS Microbiology Letters, Volume 256, Issue 1, March 2006, Pages 165–170,</ref>
Once invaded the intestine epithelial cells, Aeromonas can reach any organ via the blood. Multiple virulence factor than promote their pathogenicity.

The nonintestinal form of the disease reports symptoms such as [http://en.wikipedia.org/wiki/Sepsis septicemia], lesions of skin and soft tissues as well as [http://en.wikipedia.org/wiki/Meningitis meningitis], often ending fatally. That leads to a crucial reduction in tissue perfusion followed by fatal organ disfunction.

The ASP induced proteolysis digestion of proteins like [http://en.wikipedia.org/wiki/Kininogen kininogen], [http://en.wikipedia.org/wiki/Thrombin prothrombin], [http://en.wikipedia.org/wiki/Fibrinogen fibrinogen] or [http://en.wikipedia.org/wiki/Prekallikrein prekallikrein] at restricted sites generates fragments, expressing their own activity and therefore inducing specific physiological reactions. The [http://en.wikipedia.org/wiki/Kinin–kallikrein_system kinin system] activation, for example, reduces the blood pressure while the prothrombin system promotes plasma coagulation.

In the kallikrein/kinin system, the ASP induce the production of kinin from kininogen (low MW or high MW). The kinin also release caused edema at the infection site and shock in the circulation. The schematic representation of this action is represented : [http://www.degruyter.com/viewimg/j/bchm.2017.398.issue-10/hsz-2016-0344/hsz-2016-0344.xml?img=graphic/j_hsz-2016-0344_fig_002.jpg '''acting points of ASP in the kinin system''' ]

Experiments have been done in order to try to reduce the virulence activity of ASP. It has been demonstrated that the [http://fr.wikipedia.org/wiki/Alpha-2_macroglobuline α2-macroglobulin], a plasma protein, can limit ASP activity. This protein can bind to ASP which is also inactivated. <ref>Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin. Murakami Y et al. Biol Chem. 393(10):1193-200 (2012)</ref>

</StructureSection>

== References ==
<references/>
<scene name='82/829344/The_p-domain/1'>Text To Be Displayed</scene>

Sandbox Reserved 1091

2020-01-16T18:40:20Z

Lukas Jan Wyrwal:

{{Sandbox_ESBS_2019}}

== '''The serine protease from ''Aeromonas sobria'' : ASP ''' ==
<StructureSection load='3hjr' size='340' side='right' caption='General structure of ASP protein (with Ca2+ Binding Site and Disulfide Bridges)' scene=''>

== Generalities ==

The ''Aeromonas Sobria Serine Protease'' ASP protein is a '''serine protease''' that will cut peptide bonds after specific amino acids of a target protein. It preferentially cleaves peptide bonds that follow dibasic amino-acid residues. The kexin-like serine protease belongs to the subtilisin family ([http://en.m.wikipedia.org/wiki/Subtilase Subtilase]). The structure of ASP is similar to that of Kex2 ([[1r64]]), a protease of the subtilisin family from Saccharomyces cerevisiae. <ref>PMID:2646633</ref>

This belonging to the subtilisin serine proteases family is hypothetical. Furthermore the predicted amino acid sequence reinforces this speculation. However, the size of the ASP (MW 65 kDa) is unlike other subtilisin proteases (MW 30 kDa). Also, the amino acid residues composition is different from the family’s characteristics because ASP shows unique cysteine residues that other family members don't show. Therefore we can state that it is likely that ASP belongs to the subtilisin serine proteases family, however it remains unclear.
Moreover, ASP is the only prokaryotic memebr of the kexin-subtilisin family that is composed almost exclusively of eukaryotic proteases like Furin.<ref>Siezen RJ & Leunissen JAM (1997) Subtilase: the superfamily of subtilisin-like serine proteases. Protein Sci 6: 501–523.</ref>
ASP was shown not to be a metalloprotease because its activity is not affected by metal chelators ([http://en.wikipedia.org/wiki/Ethylenediaminetetraacetic_acid EDTA], [http://en.wikipedia.org/wiki/EGTA_(chemical) EGTA], [http://en.wikipedia.org/wiki/Phenanthroline o-phenanthroline]) or metalloprotease inhibitors ([http://en.wikipedia.org/wiki/Phosphoramidon phosphoramidon]). <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Takahisa Imamura et al. (2017)</ref>

This protein is secreted by the Anaerobic bacterium [http://en.m.wikipedia.org/wiki/Aeromonas Aeromonas Sobria], which can cause potentially lethal [http://en.m.wikipedia.org/wiki/Septic_shock septic shock]. It is a clinical syndrome of potentially fatal organ dysfunction caused by a disorder in the response to infection. In septic shock, there is a critical reduction in tissue perfusion; acute multi visceral failure, including the lungs, kidneys and liver, can be observed. <ref>http://www.msdmanuals.com/professional/critical-care-medicine/sepsis-and-septic-shock/sepsis-and-septic-shock</ref>

ASP is a '''[http://en.wikipedia.org/wiki/Sepsis sepsis]-related factor'''. It can cause several dysfunctions like by inducing vascular leakage, reducing blood pressure via the activation of the [http://en.wikipedia.org/wiki/Kinin-kallikreinsystem kinin system] or promoting human plasma coagulation through the activation of [http://fr.m.wikipedia.org/wiki/Prothrombine prothrombin]. Finally, it can cause the formation of pus and edema through the action of anaphylatoxin C5a ([[4p3a]]). Gastroenteritis, and in extreme cases deuteropathy, are the main syndrome caused by infection with ''A.sobria''.

== Maturation ==

The precursor of ASP is composed of 624 amino acids. It contains a signal peptide of 24 amino acids, a catalytic domain, similar to that of subtilisin, and a P domain.
The '''maturation of ASP''' is achieved by ORF2. This protein plays the role of an external chaperone and is necessary for the construction of the stable ASP. Indeed, ASP doesn’t contain any propeptide that is involved in the proper folding of the protein. This is a major difference with an other protein, close to ASP : Kex2 ([[1r64]]) <ref>Structural Basis for Action of the External Chaperone for a Propeptide-deficient Serine Protease from Aeromonas sobria. Kobayashi H et al. Biol. Chem. 290(17):11130-43 (2015)</ref>

The ORF2 protein is composed of 152 amino-acids coded by the orf2 gene of 456 base pairs. The N-terminal extension and the C-terminal tail of the protein are implicated in the maturation of ASP. In fact, a complex ASP-ORF2 is formed. This association requires a specific organization of ASP in the space. The <scene name='82/829344/The_p-domain/2'>P-domain</scene> of ASP doesn’t bind to ORF2 but the sixth residue from the C-terminus domain of ORF2 interacts with the non-mature ASP. In the complex, the active site of ASP is blocked. This protects the protein from degradation by others.
When the complex is formed, it moves to the extracellular space and then it dissociates. The active ASP can dissociate ORF2 and exercise its virulence activity in the cell. <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Imamura T, Murakami Y, Nitta H. Biol. Chem. 398 1055-1068 (2017)</ref>

== Secondary structure ==

The '''structure of ASP''' is very similar to that of Kex2 ([[1r64]]), but it has a unique extra-occluding region close to its active site within the subtilisin domains. This extra-occluding region is unique and it could serve as a useful target to make the development of new antisepsis drugs easier.

The '''domain structure of ASP''' consists of the propeptide, the catalytic subtilisin-like domain, and the P-domain. The ASP molecule have two mean regions: an N-terminal region extending from Gly-3 to Pro-431 and forming the <scene name='82/829344/The_subtilisin_domain/2'>subtilisin domain</scene>, and a C-terminal region extending from Leu-432 to His-595 and forming the <scene name='82/829344/The_p-domain/2'>P-domain</scene>.

Moreover, we can find three <scene name='82/829344/Calcium_binding_sites/2'>Ca2+ Binding Sites</scene> in the ASP Structure (Ca1, Ca2 and Ca3). <scene name='82/829344/Ca1_et_ca2/4'>Ca1 and Ca2</scene> are situated in the N-terminal domain, and <scene name='82/829344/Ca3/3'>Ca3</scene> is situated in the C-terminal domain. It were assigned to ASP based on electron density, counter charges, and coordination. But in contrary to Kex2 ([[1r64]]), ASP contains no Ca2+ binding sites near its catalytic site.

A schematic representation of the domains of the protein can be observed : [http://www.degruyter.com/viewimg/j/bchm.2017.398.issue-10/hsz-2016-0344/hsz-2016-0344.xml?img=graphic/j_hsz-2016-0344_fig_001.jpg '''secondary structure of ASP''']
We can see that Kex2 has the propeptide (in yellow) that is absent in ASP. The occluding subdomains in the C-terminal region of ASP are shown in dark blue.

== Domains ==

'''The Subtilisin Domain:''' It contains ten helices (alpha 1 to 10) and twelve chains (beta 1 to 10 and béta 13 to 14). The N-terminal domain of ASP seems to be like the catalytic domain of Kex2 ([[1r64]]), which is similar to those of subtilisin and other subtilisin-related proteases. This ASP catalytic site contains <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> Asp78, His115, and Ser336 residues characteristic of subtilisins. In addition, four loops (L) protrude from the N-terminal subtilisin domain of ASP : Gly3– Pro26 (<scene name='82/829344/L1/2'>L1</scene>), Asn221–Phe241 (<scene name='82/829344/L2/2'>L2</scene>), Gly300–Cys326 (<scene name='82/829344/L3/2'>L3</scene>), and Gln-377–Glu-397 (<scene name='82/829344/L4/2'>L4</scene>). L1, L2, and L3 have random coil structure, whereas L4 forms a hairpin that protrudes toward the P-domain. Moreover, two <scene name='82/829344/Disulfide_bridges/2'>disulfide bridges</scene> are formed between Cys4 and Cys24 in L1 and between Cys301 and Cys326 in L3, which stabilize those loops.

'''The P-domain:''' The core of the P-domain in ASP contains eight beta-strands (beta 16 18 23 and 26). The <scene name='82/829344/Extra_occluding_region/2'>extra occluding-region</scene> is comprised of two parts, <scene name='82/829344/Pl1/3'>pL1</scene>(Gly521–Thr525, beta 5, 6, and 12) and <scene name='82/829344/Pl2/3'>pL2</scene> (Gly557–Asn578, béta 25), and it is situated close to <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> Asp78,His115,and Ser336.

All these domains are represented schematically in the article <ref>Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor. H Kobayashi et al. J Biol Chem. 284(40): 27655–27663 (2009)</ref> : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785694/figure/F2/ '''representation 2D of ASP''']

On these figures, we can see the different domains of the protein in A and also a superposition with the Kex2. We clearly see the resemblance between both serine protease, and the extra occluding region in the C-terminal region of ASP.

== Active site ==

'''The catalytic triad:''' The [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] of ASP is composed of '''Asp78''', '''His115''' and '''Ser336'''. These amino acids are the base is the active site of the protein, where the mode of action of the serine protease takes place. A peptide can be inserted in the space of the active site. There, the amino acids of <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> will interact together and the mechanism will lead to a cut in the polypeptide.

This triad can be observed in a 2D representation of the protein : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785694/figure/F3/ '''catalytic triad of ASP''']

'''Mechanism:''' The mechanism of the reaction is the following : The histidine will react with the serine and deprotonate it. The deprotonated hydroxyl group of the serine will act as a nucleophilic species and attack the carbon from the carbonyl function on the peptide. This will lead to the formation of a tetrahedral intermediate. The regeneration of the carbonyl group will be followed by the release of one part of the peptide, with an amine group at its extremity. Then, a second tetrahedral intermediate will be formed, but with the attack of a deprotonated water molecule. In the end, the regeneration of the active site will be done with the release of the part of the peptide with a carboxyl extremity. '''The polypeptide is also cut in two parts and the target protein isn't functional anymore'''. <ref>http://fr.wikipedia.org/wiki/Fichier:Serine_protease_mechanism_by_snellios.png</ref>

A schematic representation of the mechanism with the involved amino acids can be found under the following link : [http://fr.wikipedia.org/wiki/Fichier:Serine_protease_mechanism_by_snellios.png '''mechanism of the reaction''' ]

The peptide bonds were shown to be cleaved when two basic residues were in sequence. A Lys residue at positions P1 and P2 relative to the cleavage site is prefered. If an Arg residue is at P4 position the substrate cleavage will be enhanced. <ref>Cleavage specificity of serine protease of Aeromonas sobria, a member of the kexin family of subtilases., H. Kobayashi, Okayama University, Japan,FEMS Microbiology Letters, Volume 256, Issue 1, March 2006, Pages 165–170,</ref>

== Properties ==

ASP has its highest activity at pH 7,5 and loses it after heating at 60° for 10 minutes. <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Imamura T, Murakami Y, Nitta H. Biol. Chem. 398 1055-1068 (2017)</ref>

Experiments have been done in order to establish the sensitivity of ASP to proteases. In has been found that the ASP protease activity was strongly attenuated by serine protease inhibitors ([http://en.wikipedia.org/wiki/Diisopropyl_fluorophosphate DFP], [http://fr.wikipedia.org/wiki/Fluorure_de_4-(2-aminoéthyl)benzènesulfonyle AEBSF]). Moreover, a soybean [http://en.wikipedia.org/wiki/Trypsin_inhibitor trypsin inhibitor] was shown not to block the proteolytic action of ASP itself but could inhibit the [http://en.m.wikipedia.org/wiki/Vascular_permeability vascular permeability] enhancing activity that follows after injection of ASP into epithelial cells. <ref>Physicochemical and biological properties od an extracellular serine protease od Aeromonas sobria. Ritsuko Yokoyama, Yoshio Fujii et al. Japan (2002)</ref>

This experimental finding suggests that epithelial trypsin-like proteases mediate the reaction causing enhanced vascular permeability. It is likely that ASP stimulates the secretion and maturation of epithelial trypsin proteases, thus enhancing vascular permeability. ASP could stimulate the [http://en.wikipedia.org/wiki/Bradykinin bradykinin]-releasing pathway, thus stimulating mast cells to release histamine and further enhance the vascular permeability.
Antihistaminic agents ([http://en.wikipedia.org/wiki/Diphenhydramine diphenhydramine] and [http://en.wikipedia.org/wiki/Mepyramine pyrilamine]) were shown to efficiently inhibit vascular permeability enhancing the activity of the ASP. It is very likely that the vascular permeability enhancement is related to the release of [http://en.wikipedia.org/wiki/Histamine histamine] from [http://en.wikipedia.org/wiki/Mast_cell mast cells].
Through histopathological examinations it was shown that mast cells appeared around the injection site, confirming the role of histamine as a key factor.<ref>Physicochemical and biological properties od an extracellular serine protease od Aeromonas sobria. Ritsuko Yokoyama, Yoshio Fujii et al. Japan (2002)</ref>

== Impact on human body ==

The predominant infection vector is through exposure to water harbouring ''Aeromonas sobria''. <ref>Joseph, S. W., O. P. Daily, W. S. Hunt, R. J. Seidler, D. A. Allen, and R. R. Colwell. 1979. Aeromonas primary wound infection of a diver in polluted waters. J. Clin. Microbiol. 10:46-49.</ref>
The most common form of disease is one where ''A.sobria'' pathogen adheres to the surface of the intestine causing painful diarrhea, also known as [http://en.wikipedia.org/wiki/Gastroenteritis gastroenteritis]. The enterotoxin activity of the [http://en.wikipedia.org/wiki/Hemolysin hemolysin] [http://en.wikipedia.org/wiki/Virulence_factor virulence factors] of ''A.sobria'' contributes to those symptoms. However, the mortality due to intestinal disease type of infection is low compared to the non-intestinal diseases caused by the ''A.sobria'' infection.
Pili facilitate the adherence of ''Aeromonas'' to human buccal cells. <ref>Cleavage specificity of serine protease of Aeromonas sobria, a member of the kexin family of subtilases., H. Kobayashi, Okayama University, Japan,FEMS Microbiology Letters, Volume 256, Issue 1, March 2006, Pages 165–170,</ref>
Once invaded the intestine epithelial cells, Aeromonas can reach any organ via the blood. Multiple virulence factor than promote their pathogenicity.

The nonintestinal form of the disease reports symptoms such as [http://en.wikipedia.org/wiki/Sepsis septicemia], lesions of skin and soft tissues as well as [http://en.wikipedia.org/wiki/Meningitis meningitis], often ending fatally. That leads to a crucial reduction in tissue perfusion followed by fatal organ disfunction.

The ASP induced proteolysis digestion of proteins like [http://en.wikipedia.org/wiki/Kininogen kininogen], [http://en.wikipedia.org/wiki/Thrombin prothrombin], [http://en.wikipedia.org/wiki/Fibrinogen fibrinogen] or [http://en.wikipedia.org/wiki/Prekallikrein prekallikrein] at restricted sites generates fragments, expressing their own activity and therefore inducing specific physiological reactions. The [http://en.wikipedia.org/wiki/Kinin–kallikrein_system kinin system] activation, for example, reduces the blood pressure while the prothrombin system promotes plasma coagulation.

In the kallikrein/kinin system, the ASP induce the production of kinin from kininogen (low MW or high MW). The kinin also release caused edema at the infection site and shock in the circulation. The schematic representation of this action is represented : [http://www.degruyter.com/viewimg/j/bchm.2017.398.issue-10/hsz-2016-0344/hsz-2016-0344.xml?img=graphic/j_hsz-2016-0344_fig_002.jpg '''acting points of ASP in the kinin system''' ]

Experiments have been done in order to try to reduce the virulence activity of ASP. It has been demonstrated that the [http://fr.wikipedia.org/wiki/Alpha-2_macroglobuline α2-macroglobulin], a plasma protein, can limit ASP activity. This protein can bind to ASP which is also inactivated. <ref>Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin. Murakami Y et al. Biol Chem. 393(10):1193-200 (2012)</ref>

</StructureSection>

== References ==
<references/>
<scene name='82/829344/The_p-domain/1'>Text To Be Displayed</scene>

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Lukas Jan Wyrwal:

{{Sandbox_ESBS_2019}}

== '''The serine protease from ''Aeromonas sobria'' : ASP ''' ==
<StructureSection load='3hjr' size='340' side='right' caption='General structure of ASP protein (with Ca2+ Binding Site and Disulfide Bridges)' scene=''>

== Generalities ==

The ''Aeromonas Sobria Serine Protease'' ASP protein is a '''serine protease''' that will cut peptide bonds after specific amino acids of a target protein. It preferentially cleaves peptide bonds that follow dibasic amino-acid residues. The kexin-like serine protease belongs to the subtilisin family ([http://en.m.wikipedia.org/wiki/Subtilase Subtilase]). The structure of ASP is similar to that of Kex2 ([[1r64]]), a protease of the subtilisin family from Saccharomyces cerevisiae. <ref>PMID:2646633</ref>

This belonging to the subtilisin serine proteases family is hypothetical. Furthermore the predicted amino acid sequence reinforces this speculation. However, the size of the ASP (MW 65 kDa) is unlike other subtilisin proteases (MW 30 kDa). Also, the amino acid residues composition is different from the family’s characteristics because ASP shows unique cysteine residues that other family members don't show. Therefore we can state that it is likely that ASP belongs to the subtilisin serine proteases family, however it remains unclear.
ASP was shown not to be a metalloprotease because its activity is not affected by metal chelators ([http://en.wikipedia.org/wiki/Ethylenediaminetetraacetic_acid EDTA], [http://en.wikipedia.org/wiki/EGTA_(chemical) EGTA], [http://en.wikipedia.org/wiki/Phenanthroline o-phenanthroline]) or metalloprotease inhibitors ([http://en.wikipedia.org/wiki/Phosphoramidon phosphoramidon]). <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Takahisa Imamura et al. (2017)</ref>

This protein is secreted by the Anaerobic bacterium [http://en.m.wikipedia.org/wiki/Aeromonas Aeromonas Sobria], which can cause potentially lethal [http://en.m.wikipedia.org/wiki/Septic_shock septic shock]. It is a clinical syndrome of potentially fatal organ dysfunction caused by a disorder in the response to infection. In septic shock, there is a critical reduction in tissue perfusion; acute multi visceral failure, including the lungs, kidneys and liver, can be observed. <ref>http://www.msdmanuals.com/professional/critical-care-medicine/sepsis-and-septic-shock/sepsis-and-septic-shock</ref>

ASP is a '''[http://en.wikipedia.org/wiki/Sepsis sepsis]-related factor'''. It can cause several dysfunctions like by inducing vascular leakage, reducing blood pressure via the activation of the [http://en.wikipedia.org/wiki/Kinin-kallikreinsystem kinin system] or promoting human plasma coagulation through the activation of [http://fr.m.wikipedia.org/wiki/Prothrombine prothrombin]. Finally, it can cause the formation of pus and edema through the action of anaphylatoxin C5a ([[4p3a]]). Gastroenteritis, and in extreme cases deuteropathy, are the main syndrome caused by infection with ''A.sobria''.

== Maturation ==

The precursor of ASP is composed of 624 amino acids. It contains a signal peptide of 24 amino acids, a catalytic domain, similar to that of subtilisin, and a P domain.
The '''maturation of ASP''' is achieved by ORF2. This protein plays the role of an external chaperone and is necessary for the construction of the stable ASP. Indeed, ASP doesn’t contain any propeptide that is involved in the proper folding of the protein. This is a major difference with an other protein, close to ASP : Kex2 ([[1r64]]) <ref>Structural Basis for Action of the External Chaperone for a Propeptide-deficient Serine Protease from Aeromonas sobria. Kobayashi H et al. Biol. Chem. 290(17):11130-43 (2015)</ref>

The ORF2 protein is composed of 152 amino-acids coded by the orf2 gene of 456 base pairs. The N-terminal extension and the C-terminal tail of the protein are implicated in the maturation of ASP. In fact, a complex ASP-ORF2 is formed. This association requires a specific organization of ASP in the space. The <scene name='82/829344/The_p-domain/2'>P-domain</scene> of ASP doesn’t bind to ORF2 but the sixth residue from the C-terminus domain of ORF2 interacts with the non-mature ASP. In the complex, the active site of ASP is blocked. This protects the protein from degradation by others.
When the complex is formed, it moves to the extracellular space and then it dissociates. The active ASP can dissociate ORF2 and exercise its virulence activity in the cell. <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Imamura T, Murakami Y, Nitta H. Biol. Chem. 398 1055-1068 (2017)</ref>

== Secondary structure ==

The '''structure of ASP''' is very similar to that of Kex2 ([[1r64]]), but it has a unique extra-occluding region close to its active site within the subtilisin domains. This extra-occluding region is unique and it could serve as a useful target to make the development of new antisepsis drugs easier.

The '''domain structure of ASP''' consists of the propeptide, the catalytic subtilisin-like domain, and the P-domain. The ASP molecule have two mean regions: an N-terminal region extending from Gly-3 to Pro-431 and forming the <scene name='82/829344/The_subtilisin_domain/2'>subtilisin domain</scene>, and a C-terminal region extending from Leu-432 to His-595 and forming the <scene name='82/829344/The_p-domain/2'>P-domain</scene>.

Moreover, we can find three <scene name='82/829344/Calcium_binding_sites/2'>Ca2+ Binding Sites</scene> in the ASP Structure (Ca1, Ca2 and Ca3). <scene name='82/829344/Ca1_et_ca2/4'>Ca1 and Ca2</scene> are situated in the N-terminal domain, and <scene name='82/829344/Ca3/3'>Ca3</scene> is situated in the C-terminal domain. It were assigned to ASP based on electron density, counter charges, and coordination. But in contrary to Kex2 ([[1r64]]), ASP contains no Ca2+ binding sites near its catalytic site.

A schematic representation of the domains of the protein can be observed : [http://www.degruyter.com/viewimg/j/bchm.2017.398.issue-10/hsz-2016-0344/hsz-2016-0344.xml?img=graphic/j_hsz-2016-0344_fig_001.jpg '''secondary structure of ASP''']
We can see that Kex2 has the propeptide (in yellow) that is absent in ASP. The occluding subdomains in the C-terminal region of ASP are shown in dark blue.

== Domains ==

'''The Subtilisin Domain:''' It contains ten helices (alpha 1 to 10) and twelve chains (beta 1 to 10 and béta 13 to 14). The N-terminal domain of ASP seems to be like the catalytic domain of Kex2 ([[1r64]]), which is similar to those of subtilisin and other subtilisin-related proteases. This ASP catalytic site contains <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> Asp78, His115, and Ser336 residues characteristic of subtilisins. In addition, four loops (L) protrude from the N-terminal subtilisin domain of ASP : Gly3– Pro26 (<scene name='82/829344/L1/2'>L1</scene>), Asn221–Phe241 (<scene name='82/829344/L2/2'>L2</scene>), Gly300–Cys326 (<scene name='82/829344/L3/2'>L3</scene>), and Gln-377–Glu-397 (<scene name='82/829344/L4/2'>L4</scene>). L1, L2, and L3 have random coil structure, whereas L4 forms a hairpin that protrudes toward the P-domain. Moreover, two <scene name='82/829344/Disulfide_bridges/2'>disulfide bridges</scene> are formed between Cys4 and Cys24 in L1 and between Cys301 and Cys326 in L3, which stabilize those loops.

'''The P-domain:''' The core of the P-domain in ASP contains eight beta-strands (beta 16 18 23 and 26). The <scene name='82/829344/Extra_occluding_region/2'>extra occluding-region</scene> is comprised of two parts, <scene name='82/829344/Pl1/3'>pL1</scene>(Gly521–Thr525, beta 5, 6, and 12) and <scene name='82/829344/Pl2/3'>pL2</scene> (Gly557–Asn578, béta 25), and it is situated close to <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> Asp78,His115,and Ser336.

All these domains are represented schematically in the article <ref>Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor. H Kobayashi et al. J Biol Chem. 284(40): 27655–27663 (2009)</ref> : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785694/figure/F2/ '''representation 2D of ASP''']

On these figures, we can see the different domains of the protein in A and also a superposition with the Kex2. We clearly see the resemblance between both serine protease, and the extra occluding region in the C-terminal region of ASP.

== Active site ==

'''The catalytic triad:''' The [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] of ASP is composed of '''Asp78''', '''His115''' and '''Ser336'''. These amino acids are the base is the active site of the protein, where the mode of action of the serine protease takes place. A peptide can be inserted in the space of the active site. There, the amino acids of <scene name='82/829344/Catalytic_triad/2'>the catalytic triad</scene> will interact together and the mechanism will lead to a cut in the polypeptide.

This triad can be observed in a 2D representation of the protein : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785694/figure/F3/ '''catalytic triad of ASP''']

'''Mechanism:''' The mechanism of the reaction is the following : The histidine will react with the serine and deprotonate it. The deprotonated hydroxyl group of the serine will act as a nucleophilic species and attack the carbon from the carbonyl function on the peptide. This will lead to the formation of a tetrahedral intermediate. The regeneration of the carbonyl group will be followed by the release of one part of the peptide, with an amine group at its extremity. Then, a second tetrahedral intermediate will be formed, but with the attack of a deprotonated water molecule. In the end, the regeneration of the active site will be done with the release of the part of the peptide with a carboxyl extremity. '''The polypeptide is also cut in two parts and the target protein isn't functional anymore'''. <ref>http://fr.wikipedia.org/wiki/Fichier:Serine_protease_mechanism_by_snellios.png</ref>

A schematic representation of the mechanism with the involved amino acids can be found under the following link : [http://fr.wikipedia.org/wiki/Fichier:Serine_protease_mechanism_by_snellios.png '''mechanism of the reaction''' ]

The peptide bonds were shown to be cleaved when two basic residues were in sequence. A Lys residue at positions P1 and P2 relative to the cleavage site is prefered. If an Arg residue is at P4 position the substrate cleavage will be enhanced. <ref>Cleavage specificity of serine protease of Aeromonas sobria, a member of the kexin family of subtilases. H. Kobayashi</ref>

== Properties ==

ASP has its highest activity at pH 7,5 and loses it after heating at 60° for 10 minutes. <ref>Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities. Imamura T, Murakami Y, Nitta H. Biol. Chem. 398 1055-1068 (2017)</ref>

Experiments have been done in order to establish the sensitivity of ASP to proteases. In has been found that the ASP protease activity was strongly attenuated by serine protease inhibitors ([http://en.wikipedia.org/wiki/Diisopropyl_fluorophosphate DFP], [http://fr.wikipedia.org/wiki/Fluorure_de_4-(2-aminoéthyl)benzènesulfonyle AEBSF]). Moreover, a soybean [http://en.wikipedia.org/wiki/Trypsin_inhibitor trypsin inhibitor] was shown not to block the proteolytic action of ASP itself but could inhibit the [http://en.m.wikipedia.org/wiki/Vascular_permeability vascular permeability] enhancing activity that follows after injection of ASP into epithelial cells. <ref>Physicochemical and biological properties od an extracellular serine protease od Aeromonas sobria. Ritsuko Yokoyama, Yoshio Fujii et al. Japan (2002)</ref>

This experimental finding suggests that epithelial trypsin-like proteases mediate the reaction causing enhanced vascular permeability. It is likely that ASP stimulates the secretion and maturation of epithelial trypsin proteases, thus enhancing vascular permeability. ASP could stimulate the [http://en.wikipedia.org/wiki/Bradykinin bradykinin]-releasing pathway, thus stimulating mast cells to release histamine and further enhance the vascular permeability.
Antihistaminic agents ([http://en.wikipedia.org/wiki/Diphenhydramine diphenhydramine] and [http://en.wikipedia.org/wiki/Mepyramine pyrilamine]) were shown to efficiently inhibit vascular permeability enhancing the activity of the ASP. It is very likely that the vascular permeability enhancement is related to the release of [http://en.wikipedia.org/wiki/Histamine histamine] from [http://en.wikipedia.org/wiki/Mast_cell mast cells].
Through histopathological examinations it was shown that mast cells appeared around the injection site, confirming the role of histamine as a key factor.

== Impact on human body ==

The most common form of disease is one where ''A.sobria'' pathogen adheres to the surface of the intestine causing painful diarrhea, also known as [http://en.wikipedia.org/wiki/Gastroenteritis gastroenteritis]. The enterotoxin activity of the [http://en.wikipedia.org/wiki/Hemolysin hemolysin] [http://en.wikipedia.org/wiki/Virulence_factor virulence factors] of ''A.sobria'' contributes to those symptoms. However, the mortality due to intestinal disease type of infection is low compared to the non-intestinal diseases caused by the ''A.sobria'' infection.
Once invaded the intestine epithelial cells, Aeromonas can reach any organ via the blood. Multiple virulence factor than promote their pathogenicity.

The nonintestinal form of the disease reports symptoms such as [http://en.wikipedia.org/wiki/Sepsis septicemia], lesions of skin and soft tissues as well as [http://en.wikipedia.org/wiki/Meningitis meningitis], often ending fatally. That leads to a crucial reduction in tissue perfusion followed by fatal organ disfunction.

The ASP induced proteolysis digestion of proteins like [http://en.wikipedia.org/wiki/Kininogen kininogen], [http://en.wikipedia.org/wiki/Thrombin prothrombin], [http://en.wikipedia.org/wiki/Fibrinogen fibrinogen] or [http://en.wikipedia.org/wiki/Prekallikrein prekallikrein] at restricted sites generates fragments, expressing their own activity and therefore inducing specific physiological reactions. The [http://en.wikipedia.org/wiki/Kinin–kallikrein_system kinin system] activation, for example, reduces the blood pressure while the prothrombin system promotes plasma coagulation.

In the kallikrein/kinin system, the ASP induce the production of kinin from kininogen (low MW or high MW). The kinin also release caused edema at the infection site and shock in the circulation. The schematic representation of this action is represented : [http://www.degruyter.com/viewimg/j/bchm.2017.398.issue-10/hsz-2016-0344/hsz-2016-0344.xml?img=graphic/j_hsz-2016-0344_fig_002.jpg '''acting points of ASP in the kinin system''' ]

Experiments have been done in order to try to reduce the virulence activity of ASP. It has been demonstrated that the [http://fr.wikipedia.org/wiki/Alpha-2_macroglobuline α2-macroglobulin], a plasma protein, can limit ASP activity. This protein can bind to ASP which is also inactivated. <ref>Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin. Murakami Y et al. Biol Chem. 393(10):1193-200 (2012)</ref>

</StructureSection>

== References ==
<references/>
<scene name='82/829344/The_p-domain/1'>Text To Be Displayed</scene>

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Lukas Jan Wyrwal:

{{Sandbox_ESBS_2019}}
== The serine protease from ''Aeromonas sobria'' ==
<StructureSection load='3hjr' size='340' side='right' caption='Caption for this structure' scene=''>

== Generalities ==

The ''Aeromonas Sobria Serine Protease'' ASP protein is a '''serine protease''' that will cut peptide bonds after specific amino acids of a target protein. It preferentially cleaves peptide bonds that follow dibasic amino-acid residues. The kexin-like serine protease belongs to the subtilisin family ([http://en.m.wikipedia.org/wiki/Subtilase Subtilase]). The structure of ASP is similar to that of ''Kex2'' <ref>PMID:2646633</ref> ([[1r64]]), a protease of the subtilisin family, but ASP has a unique extra occluding region close to its active site.

This protein is secreted by the Anaerobic bacterium [http://en.m.wikipedia.org/wiki/Aeromonas Aeromonas Sobria], which can cause potentially lethal septic shock. [http://www.mdsmanuals.com Septic Shock] is a clinical syndrome of potentially fatal organ dysfunction caused by a disorder in the response to infection. In septic shock, there is a critical reduction in tissue perfusion; acute multivisceral failure, including the lungs, kidneys and liver, can be observed.

ASP is a '''[http://en.m.wikipedia.org/wiki/Sepsis sepsis]-related factor'''. It can cause several dysfunction like by inducing vascular leakage, reducing blood pressure via the activation of the [http://en.m.wikipedia.org/wiki/Kinin-kallikreinsystem kinin system] or promoting human plasma coagulation through the activation of [http://fr.m.wikipedia.org/wiki/Prothrombine prothrombin]. Finally it can causes the formation of pus and edema through the action of anaphylatoxin C5a ([[4p3a]]). Gastroenteritis, and in extreme cases deuteropathy, are the main syndrome caused by infection with ''A.sobria''.

The '''maturation of ASP''' is achieved by ORF2. This protein plays the role of an external chaperone and is necessary for the construction of the stable ASP. Indeed, ASP doesn’t contain a propeptide (such as Kex2) that is involved in the proper folding of the protein.

Phrase dans maturation à reformuler … “For maturation of ASP, the first 24 residues of the propeptide are cleaved and although a functional P-domain is reportedly necessary for maturation of the substitution domain in kexins”

== Secondary structure ==

The '''structure of ASP''' is very similar to that of ''Kex2'' ([[1r64]]), but it has a unique extra-occluding region close to its active site within the subtilisin domains. This extra-occluding region is unique and it could serve as a useful target to make the development of new antisepsis drugs easier.

The '''domain structure of ASP''' consists of the propeptide, the catalytic subtilisin-like domain, and the P-domain. The ASP molecule have two mean regions: an N-terminal region extending from Gly-3 to Pro-431 and forming '''the subtilisin domain''', and a C-terminal region extending from Leu-432 to His-595 and forming '''the P-domain'''.

Moreover, we can find three '''Ca2+ Binding Sites''' in the ASP Structure (Ca1, Ca2 and Ca3). Ca1 and Ca2 are situated in the N-terminal domain, and Ca3 is situated in the C-terminal domain. It were assigned to ASP based on electron density, counter charges, and coordination. But in contrary to ''Kex2'', ASP contains no Ca2 binding sites near its catalytic site. Those Ca2+ binding Site are important because ...

== Domains ==

'''The Subtilisin Domain:''' It contains 10 helices (alpha 1 to 10) and twelve chains (béta 1 to 10 and béta 13 to 14). The N-terminal domain of ASP seems to be like the catalytic domain of ''Kex2'', which is similar to those of subtilisin and other subtilisin-related proteases. This ASP catalytic site contains the catalytic Asp-78, His-115, and Ser-336 residues characteristic of subtilisins. In addition, 4 loops (L) protrude from the N-terminal subtilisin domain of ASP: Gly-3– Pro-26 (L1), Asn-221–Phe-241 (L2), Gly-300–Cys-326 (L3), and Gln-377–Glu-397 (L4). L1, L2, and L3 have random coil structure, whereas L4 forms a hairpin that protrudes toward the P-domain. Moreover, two disulfide bridges are formed between Cys-4 and Cys-24 in L1 and between Cys-301 and Cys-326 in L3, which stabilize those loops.

'''The P-domain:''' The core of the P-domain in ASP contains 8 béta-strands (béta 16 18 23 and 26). The extra occluding-region is comprised of two parts, pL1(Gly 521–Thr 525, béta 5, 6, and 12) and pL2 (Gly-557–Asn-578, béta 25), and it is situated close to the catalytic triad Asp-78,His-115,and Ser-336.

https://www.degruyter.com/view/j/bchm.2017.398.issue-10/hsz-2016-0344/graphic/j_hsz-2016-0344_fig_001.jpg

== Active site ==

'''The catalytic triad:''' The [https://en.wikipedia.org/wiki/ catalytic triad] of ASP is composed of Asp78, His115 and Ser336. These amino acids are the base is the active site of the protein, where the mode of action of the serine protease takes place.

A peptide can be inserted in the space of the active site. There, the amino acids of the catalytic triad will interact together and the mechanism will lead to a cut in the polypeptide.

'''Mechanism:''' The mechanism is the following: The histidine will react with the serine and deprotonate it. '''The deprotonated hydroxyl group of the serine will act as a nucleophilic species''' and attack the carbon from the carbonyl function on the peptide. This will lead to the formation of a tetrahedral intermediate. Then, a second tetrahedral intermediate will be formed, but with the attack of a deprotonated water molecule. At the end, the regeneration of the active site will be done with the release of the peptide cut in two parts.

https://upload.wikimedia.org/wikipedia/commons/1/17/Serine_protease_mechanism_by_snellios.png

== Classification and properties ==

Performed experiments aimed to study the classification of ASP through inhibition, as well as the ability to enhance vascular permeability in dorsal skin tissue of rodents (Wistar rat).

ASP was shown not to be a metallo-protease, because its activity is not affected by metal chelators (EDTA, EGTA, o-phenantroline) or metallo-protease inhibitors (phosphoramidon).

The ASP protease activity was strongly attenuated by serine protease inhibitors (DFP, AEBSEF) suggesting a hypothetical belonging to the subtilisin serine proteases family. Furthermore the predicted amino acid sequence reinforces this speculation. However, the size of the ASP (MW 65000) is unlike other subtilisin proteases (MW 30000). Also the amino acid residues composition is different from the family’s characteristics because ASP shows unique cysteine residues that other family members don t show. Therefore we can state that it is likely that ASP belongs to the subtilisin serine proteases family, however it remains unclear.

A soybean trypsin inhibitor was shown not to block the proteolytic action of ASP itself, but could inhibit the vascular permeability enhancing activity that follows after injection of ASP into epithelial cells.
This experimental finding suggests that epithelial trypsin like proteases mediate the reaction causing enhanced vascular permeability. It is likely that ASP stimulates the secretion and maturation of epithelial trypsin proteases, thus enhancing the vascular permeability. ASP could stimulate the bradykinin-releasing pathway, thus stimulating mast cells to release histamine and further enhance the vascular permeability.
Antihistaminic agents (diphenhydramine and pyrilamine) were shown to efficiently inhibit the vascular permeability enhancing activity of the ASP. It is very likely that the vascular permeability enhancement is related to the release of histamine from mast cells.
Through histopathological examinations it was shown that mast cells appeared around the injection site, confirming the role of histamine as a key factor.

== Impact on human body ==

The most common form of desease is one where A sobria pathogen adheres to the surface of the intestine causing painful diarrhoea, also known as gastroenteritis. The enterotoxic activity of the Hemolysin virulence factor of A. sobria contributes to those symptoms. However, the mortality due to intestinal disease type of infection is low compared to the non-intestinal diseases caused by A. Sobria infection.
Once invaded the intestine epithel cells, Aeromonas can reach any organ via the blood. Multiple virulence factors than promote their pathogenicity.

The nonintestinal form of the disease reports symptoms such as septicaemia, lesions of skin and soft tissues as well as meningitis, often ending fatally.
leading to crucial reduction in tissue perfusion followed by fatal organ disfunction.

The ASP induced proteolysis digestion of proteins like kininogen, prothrombin, fibrinogen or prekallikrein at restricted sites generates fragments, expressing their own activity and therefore inducing specific physiological reactions. The kinin system activation for example reduces the blood pressure while the prothrombin system promotes plasma coagulation.
...

</StructureSection>

== References ==
<references/>

Sandbox Reserved 1091

2020-01-13T14:21:23Z

Lukas Jan Wyrwal:

{{Sandbox_ESBS_2019}}
== The serine protease from ''Aeromonas sobria'' ==
<StructureSection load='3hjr' size='340' side='right' caption='Caption for this structure' scene=''>

== Generalities ==

The ''Aeromonas Sobria Serine Protease'' ASP protein is a '''serine protease''' that will cut peptide bonds after specific amino acids of a target protein. It preferentially cleaves peptide bonds that follow dibasic amino-acid residues. The kexin-like serine protease belongs to the subtilisin family ([http://en.m.wikipedia.org/wiki/Subtilase Subtilase]). The structure of ASP is similar to that of ''Kex2'' <ref>PMID:2646633</ref> ([[1r64]]), a protease of the subtilisin family, but ASP has a unique extra occluding region close to its active site.

This protein is secreted by the Anaerobic bacterium [http://en.m.wikipedia.org/wiki/Aeromonas Aeromonas Sobria], which can cause potentially lethal septic shock. [http://www.mdsmanuals.com Septic Shock] is a clinical syndrome of potentially fatal organ dysfunction caused by a disorder in the response to infection. In septic shock, there is a critical reduction in tissue perfusion; acute multivisceral failure, including the lungs, kidneys and liver, can be observed.

ASP is a '''[http://en.m.wikipedia.org/wiki/Sepsis sepsis]-related factor'''. It can cause several dysfunction like by inducing vascular leakage, reducing blood pressure via the activation of the [http://en.m.wikipedia.org/wiki/Kinin-kallikreinsystem kinin system] or promoting human plasma coagulation through the activation of [http://fr.m.wikipedia.org/wiki/Prothrombine prothrombin]. Finally it can causes the formation of pus and edema through the action of anaphylatoxin C5a ([[4p3a]]). Gastroenteritis, and in extreme cases deuteropathy, are the main syndrome caused by infection with ''A.sobria''.

The '''maturation of ASP''' is achieved by ORF2. This protein plays the role of an external chaperone and is necessary for the construction of the stable ASP. Indeed, ASP doesn’t contain a propeptide (such as Kex2) that is involved in the proper folding of the protein.

Phrase dans maturation à reformuler … “For maturation of ASP, the first 24 residues of the propeptide are cleaved and although a functional P-domain is reportedly necessary for maturation of the substitution domain in kexins”

== Secondary structure ==

The '''structure of ASP''' is very similar to that of ''Kex2'' ([[1r64]]), but it has a unique extra-occluding region close to its active site within the subtilisin domains. This extra-occluding region is unique and it could serve as a useful target to make the development of new antisepsis drugs easier.

The '''domain structure of ASP''' consists of the propeptide, the catalytic subtilisin-like domain, and the P-domain. The ASP molecule have two mean regions: an N-terminal region extending from Gly-3 to Pro-431 and forming '''the subtilisin domain''', and a C-terminal region extending from Leu-432 to His-595 and forming '''the P-domain'''.

Moreover, we can find three '''Ca2+ Binding Sites''' in the ASP Structure (Ca1, Ca2 and Ca3). Ca1 and Ca2 are situated in the N-terminal domain, and Ca3 is situated in the C-terminal domain. It were assigned to ASP based on electron density, counter charges, and coordination. But in contrary to ''Kex2'', ASP contains no Ca2 binding sites near its catalytic site. Those Ca2+ binding Site are important because ...

== Domains ==

'''The Subtilisin Domain:''' It contains 10 helices (alpha 1 to 10) and twelve chains (béta 1 to 10 and béta 13 to 14). The N-terminal domain of ASP seems to be like the catalytic domain of ''Kex2'', which is similar to those of subtilisin and other subtilisin-related proteases. This ASP catalytic site contains the catalytic Asp-78, His-115, and Ser-336 residues characteristic of subtilisins. In addition, 4 loops (L) protrude from the N-terminal subtilisin domain of ASP: Gly-3– Pro-26 (L1), Asn-221–Phe-241 (L2), Gly-300–Cys-326 (L3), and Gln-377–Glu-397 (L4). L1, L2, and L3 have random coil structure, whereas L4 forms a hairpin that protrudes toward the P-domain. Moreover, two disulfide bridges are formed between Cys-4 and Cys-24 in L1 and between Cys-301 and Cys-326 in L3, which stabilize those loops.

'''The P-domain:''' The core of the P-domain in ASP contains 8 béta-strands (béta 16 18 23 and 26). The extra occluding-region is comprised of two parts, pL1(Gly 521–Thr 525, béta 5, 6, and 12) and pL2 (Gly-557–Asn-578, béta 25), and it is situated close to the catalytic triad Asp-78,His-115,and Ser-336.

https://www.degruyter.com/view/j/bchm.2017.398.issue-10/hsz-2016-0344/graphic/j_hsz-2016-0344_fig_001.jpg

== Active site ==

'''The catalytic triad:''' The [https://en.wikipedia.org/wiki/ catalytic triad] of ASP is composed of Asp78, His115 and Ser336. These amino acids are the base is the active site of the protein, where the mode of action of the serine protease takes place.

A peptide can be inserted in the space of the active site. There, the amino acids of the catalytic triad will interact together and the mechanism will lead to a cut in the polypeptide.

'''Mechanism:''' The mechanism is the following: The histidine will react with the serine and deprotonate it. '''The deprotonated hydroxyl group of the serine will act as a nucleophilic species''' and attack the carbon from the carbonyl function on the peptide. This will lead to the formation of a tetrahedral intermediate. Then, a second tetrahedral intermediate will be formed, but with the attack of a deprotonated water molecule. At the end, the regeneration of the active site will be done with the release of the peptide cut in two parts.

https://upload.wikimedia.org/wikipedia/commons/1/17/Serine_protease_mechanism_by_snellios.png

== Classification and properties ==

Performed experiments aimed to study the classification of ASP through inhibition, as well as the ability to enhance vascular permeability in dorsal skin tissue of rodents (Wistar rat).

ASP was shown not to be a metallo-protease, because its activity is not affected by metal chelators (EDTA, EGTA, o-phenantroline) or metallo-protease inhibitors (phosphoramidon).

The ASP protease activity was strongly attenuated by serine protease inhibitors (DFP, AEBSEF) suggesting a hypothetical belonging to the subtilisin serine proteases family. Furthermore the predicted amino acid sequence reinforces this speculation. However, the size of the ASP (MW 65000) is unlike other subtilisin proteases (MW 30000). Also the amino acid residues composition is different from the family’s characteristics because ASP shows unique cysteine residues that other family members don t show. Therefore we can state that it is likely that ASP belongs to the subtilisin serine proteases family, however it remains unclear.

A soybean trypsin inhibitor was shown not to block the proteolytic action of ASP itself, but could inhibit the vascular permeability enhancing activity that follows after injection of ASP into epithelial cells.
This experimental finding suggests that epithelial trypsin like proteases mediate the reaction causing enhanced vascular permeability. It is likely that ASP stimulates the secretion and maturation of epithelial trypsin proteases, thus enhancing the vascular permeability. ASP could stimulate the bradykinin-releasing pathway, thus stimulating mast cells to release histamine and further enhance the vascular permeability.
Antihistaminic agents (diphenhydramine and pyrilamine) were shown to efficiently inhibit the vascular permeability enhancing activity of the ASP. It is very likely that the vascular permeability enhancement is related to the release of histamine from mast cells.
Through histopathological examinations it was shown that mast cells appeared around the injection site, confirming the role of histamine as a key factor.

</StructureSection>

== References ==
<references/>

Sandbox Reserved 1501

2019-01-10T12:41:59Z

Lukas Jan Wyrwal:

{{Sandbox_Reserved_ESBS}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function ==

Hallo

== Disease ==
sssssssjksdnfisdjsamkxksmcsa

== Relevance ==

== Structural highlights ==

qkjfghds

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox Reserved 1501

2019-01-10T12:41:18Z

Lukas Jan Wyrwal:

{{Sandbox_Reserved_ESBS}}
==Your Heading Here (maybe something like 'Structure')==
<StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''>
This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.

== Function ==

Hallo

== Disease ==

jswdnsjikefdflklnwe
gergg4we4

== Relevance ==

== Structural highlights ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>