Proteopedia - User contributions [en]

Molecular Playground/HIV Tat

2014-12-11T01:45:30Z

Coralie Backlund:

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground].

== '''Introduction''' ==

'''HIV-1 TAT''', or simply Tat, is a human immunodeficiency virus (HIV) gene that regulates transcription of HIV dsRNA.<ref>Sodroski ''et al. Science.'' '''1985'''. 227, 171-173. [http://www.jstor.org/stable/1695050] http://www.jstor.org/stable/1695050</ref> The protein is modeled here <scene name='47/477029/Hiv_tat3/3'>as a cartoon</scene> to highlight the lack of an alpha helix or a beta sheet. TAT, which stands for trans-activator of transcription, contains 86 amino acid residues in its sequence.<ref>Arya ''et al. Science'' '''1985'''. 229, 69-73.</ref><ref>Sodroski ''et al. Science.'' '''1985'''. 229, 74-77.</ref> <Structure load='1TIV' size='500' frame='true' align='right' caption='Proteins that contain protein transduction domains. When the background is black, the protein modeled is HIV-1 TAT and when the background is white, the protein modeled is Antennapedia.' scene='Brittany_deRonde/Sandbox_1/Hiv_tat/2' /> The protein is modeled such that hydrophobic residues are pink and polar residues are grey. It is released by HIV infected cells in order to enhance replication of the virus.<ref>Dayton ''et al. Cell.'' '''1986'''. 44, 941-947.</ref><ref>Fisher ''et al. Nature.'' '''1986'''. 320, 367-371.</ref> Nanomolar concentrations of TAT have been reported in the blood of HIV-1 infected people.<ref>Xiao ''et al. Proc. Natl. Acad. Sci. USA.'' '''2000'''. 97, 11466-11471.</ref> When the protein enters non-infected cells, the transcription efficiency increases. It is estimated that transcription levels are 10 to 100 fold higher during the transcription elongation stage when compared to normal basal transcription.<ref>Ensoli ''et al. Nature.'' '''1990'''. 345, 84-86.</ref> Green and Lowenstein, and Frankel and Pabo independently demonstrated in 1988 that Tat had the ability to cross cellular membranes and initiate transcription of HIV dsRNA.<ref>Green ''et al. Cell.'' '''1988'''. 55, 1179-1188.</ref><ref>Frankel ''et al. Cell.'' '''1988'''. 55, 1189-1193.</ref> These were the first known reports of a '''cell penetrating peptide (CPP)'''.

== '''Cell Penetrating Peptides''' ==

'''Cell penetrating peptides (CPPs)''' are proteins with the ability to cross cellular membranes and facilitate the uptake of various cargo, such as small molecules, protiens, antibodies, siRNA, and small DNA fragments.<ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref> Such cargoes can be associated with CPPs via covalent or non-covalent interactions. HIV Tat is considered a CPP because it contains a protein transduction domain (PTD). PTDs are cation-rich sequences of 10-30 residues, usually containing several Arginine and/or Lysine residues.<ref>Wender ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 452-472.</ref>

[[Image:Arginine1.jpg]] [[Image:Lysine1.jpg]]

These sequences improve interactions between CPPs and cellular membranes and can help them enter cells. The PTD sequence in HIV TAT is YGRKKRRQRRR (amino acid <scene name='Brittany_deRonde/Sandbox_1/Hiv_tat/3'>residues 47-57</scene>). The PTD is rich in <scene name='Molecular_Playground/HIV_Tat/Hiv_tat2/1'>Arginine and Lysine residues.</scene> (Arginine and Lysine ) This <scene name='47/477029/Hiv_tat2/2'>11 amino-acid sequence</scene> is now referred to as the TAT peptide, and has been shown to have improved cellular uptake compared to HIV TAT.

Since the discovery of HIV TAT, other Arginine-rich, natural CPPs have been discovered including, penetratin from Drosophila antennapedia protein <scene name='47/477029/Antennapedia/1'>(model of antennapedia)</scene>, sweet arrow peptide (SAP), HIV-1 Rev, flock house virus (FHV) coat, brome mosaic virus (BMV) Gag, human T-cell lymphotrophic virus (HTLV)-II Rex, and the nuclear localization signal (NLS) from nucleoplasmin. <ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref><ref>Prochiantz ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 448-451.</ref> The PTD sequences for these proteins can be found in the table below.

[[Image:Arginine_Rich_Peptides.png]]

== '''TAT-mediated Transduction''' ==

The PTD in HIV TAT is highly cationic. When charged residues (Arginine or Lysine) are replaced with neutral residues (Alanine), cellular uptake decreases. It is likely that the cationic charge is necessary for electrostatic interactions with components of the cellular membrane, such as lipid head groups and proteoglycans.<ref>Ziegler ''et al. Biochemistry'' '''2003'''. 42, 9185-9194.</ref><ref>Goncalves ''et al. Biochemistry.'' '''2005'''. 44, 2692-2702.</ref> Other interactions may also play a role in interactions with the cellular membrane, such as hydrophobic and non-covalent interactions.<ref>Ziegler ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 580-597.</ref> Although cellular uptake is not directly dependent upon architecture (branched vs. linear), it is dependent upon the number of Arginine residues. Polymers with less than five arginine residues are unable to enter cells.<ref>Wender ''et al. Proc. Natl. Acad. Sci.'' '''2000'''. 97, 13003-13008.</ref> As the number of residues increases, cellular uptake improves up until 15 residues where polymers can enter cells but with hindered efficiencies.<ref>Mitchell ''et al. J. Pept. Res.'' '''2000'''. 56, 318-325.</ref><ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref> The exact mechanism of cellular uptake is still highly debated in the literature, but a couple of the proposed methods are discussed below.

==Transduction==
Early studies indicated that HIV TAT primarily entered cells through transduction, meaning that the protein directly crossed the cell membranes. However, for most of these studies, cells were fixed with formaldehyde prior to sample analysis. In 2003, it was discovered that by fixing cells prior to analysis, it permeablized the cell membranes and resulted in artificially high cellular uptake values. Fixed cells showed higher concentrations of TAT in nuclei compared to non-fixed cells where it was localized in the cytoplasm. <ref>Richard ''et al. J. Biol. Chem.'' '''2003'''. 278, 585-590.</ref> Studies also showed that FACS could not tell the difference between internalized cells and those proteins that were only bound to the cellular membranes. Cellular uptake studies performed at 4°C and under conditions of ATP depletion show that a small percentage of CPPs can enter cells and also suggests that a majority of the protein enters cells through energy dependent pathways.<ref>Drin ''et al. J. Biol. Chem.'' '''2003'''. 276, 31192-31201.</ref> Some groups have also studied transduction of Tat using model vesicles. <ref>Ziegler ''et al. Biochemistry.'' '''2003'''. 278, 9185-9194.</ref>

==Endocytosis==
Another proposed mechanism for cellular uptake is endocytosis, specifically macropinocytosis. This form of fluid phase endocytosis is less known that other types (phagocytosis, clathrin, and coated pit), it is believed to occur in all cell types and enable the uptake of large molecules. <ref>Gump ''et al. Trends in Molecular Medicine.'' '''2007'''. 13, 443-448.</ref> During this process, actin protrusions fold around molecules and the surrounding medium and enable cellular uptake.

== '''Tew Group Research Interests''' ==
As part of the [http://www.pse.umass.edu/gtew/index.html Tew Research Group] in the [http://www.pse.umass.edu Department of Polymer Science and Engineering] at the University of Massachusetts, Amherst, We are interested in the design of TAT-inspired CPPs for drug and gene delivery. Using functionalized oxanorbornene derivatives as monomers (as shown below), ring opening metathesis polymerization (ROMP) is performed using Grubbs' 3rd generation catalyst to synthesize cationic CPP mimics (CPPMs). Hydrophobicity, charge, aromaticity, and pi-electronics of the monomer structures can be optimized to achieve better cellular uptake and cell viability by changing R1 and R2.

[[Image:norbornenederivatives.png]]

== '''Acknowledgments''' ==
The Tew research group is gratefully acknowledged for their contributions to the design and content of this page.

==3D structures of Tat protein==

[[Tat protein]]

== '''References''' ==
{{Reflist}}

CBI Molecules

2014-12-10T19:06:49Z

Coralie Backlund:

These are molecules under study by members of the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program].
Many of the molecules we study are featured at the [http://www.molecularplayground.org/ Molecular Playground] (see also [[Molecular Playground|Molecular Playground in Proteopedia]]). Follow the links below to read nontechnical descriptions, in Proteopedia, of these molecules.

UMass CBI Members, add your molecules to the list (which is alphabetical by CBI research mentor); follow the instructions below the list.

Fall 2014: CBI Molecules are due 12/3/14 and should be added at the TOP of the list from your lab. Label it as "in progress" until you are finished, and then as "New 2014" or "Revised 2014". For those editing an existing CBI Molecule, start by making a copy of that entry (leaving the original intact) so that it retains the full author list of all authors that contributed (or include credits and a link to the original page and authors if it does not).
Follow the instructions at the bottom of this page.

==Molecules==
'''** Designates CBI Molecules Featured on [http://www.umass.edu/cbi/ CBI website]'''

[http://chamberslab.com/wp/ Chambers Lab]

: '''[[Molecular Playground/Glutamate Receptor]]''', Amanda Hussey, Steve McCarron, Rosie Combs-Bachmann, Mariel Feliciano

[http://www.chem.umass.edu/~chenlab/index.HTML Chen Lab]

: '''[[Molecular Playground/ClyA]]''', Bib Yang
: '''[[Molecular Playground/OmpG]]''', Christina Chisholm

[http://openwetware.org/wiki/Chien Chien Lab]

: '''[[Molecular Playground/ClpP]]''', Lisa Hernandez, Rob Vass '''New 2014'''
: '''[[Molecular Playground/Homo-dimeric RcdA]]''', Kamal K. Joshi
: **'''[[Molecular Playground/Hexameric ClpX]]''', Joanne Lau '''Best CBI Molecule 2011'''
: '''[[Molecular Playground/DNA replication initiator DnaA]]''', Jing Liu

Farkas Lab
: '''[[Molecular Playground/CLOCK:BMAL1 heterodimer complex]]''', Hui-Hsien Lin, Joseph Hardie, Michael Mingroni '''New 2014'''

Forbes Lab

: '''[[Molecular Playground/CheR]]''', Miaomin Zhang
: '''[[Molecular Playground/RBP]]''', Jan Panteli
: '''[[Molecular Playground/TRAIL]]''', Charley Swofford

[http://www.biochem.umass.edu/garman/index.html Garman Lab]

: '''[[Molecular Playground/Human Protective Protein Cathepsin A]]''', Yadilette Rivera-Colon
: '''[[Molecular Playground/Human PPCA]]''', Nilima Kolli

[http://people.chem.umass.edu/gieraschlab/ Gierasch Lab]

: '''[[Molecular_Playground/DnaK]]''', Joseph Tilitsky, New 2014
: '''[[CRABP I ( Cellular Retinoic Acid Binding Protein )|Molecular Playground/CRABP I]]''', Kristine Faye Pobre, Mylene Ferrolino,Mangai Periasamy '''Best Overall CBI Molecule 2010'''
: '''[[Molecular Playground/CRABP I (Cellular Retinoic Acid Binding Protein)]]''', Gustavo Elberto Epalza Sanchez
: **'''[[Molecular_Playground/DHFR]]''', Karan Hingorani '''Best CBI Molecule 2012'''

[http://people.chem.umass.edu/jhardy/ Hardy Lab]

: '''[[Molecular Playground/Executioner Caspase-7]]''', Derek MacPherson, Maureen E. Hill
: '''[[Molecular Playground/Caspase-3 Regulatory Mechanisms]]''', Scott Eron, Bay Serrano, Yunlong Zhao
: '''[[Molecular Playground/Caspase-6 and neurodegeneration]]''', Kevin Buadlart Dagbay
: '''[[Molecular Playground/Caspase-7 Dynamics]]'''
: '''[[Molecular Playground/Caspase-9 Regulation]]''', Kristen Huber
: '''[[Molecular Playground/Caspase-6 (new)]]''', Elih Velazquez
: '''[[Molecular Playground/Influenza A M2 transmembrane domain]]''', Samantha Nicholls
: '''[[Molecular Playground/Dengue Virus Protease]]''', Muslum Yildiz

Hebert Lab

: '''[[Molecular_Playground/Hsp70-Hsp90]]''', Jill Graham
: '''[[Molecular_Playground/ERDj5]]''', Lydia Lamriben
: '''[[Molecular Playground/Saposin C]]''', Abla Tannous
: '''[[Molecular_Playground/ERMan1]]''', Johan Sunryd

[http://people.biochem.umass.edu/aheuck/aph.html Heuck Lab]

: '''[[Molecular_Playground/Pcr H]]''', Fabian Romano, Carolina Morell-Pérez, Yuzhou Tang

[http://people.chem.umass.edu/mholden/ Holden Lab]

: '''[[Molecular Playground/Protective Antigen]]''', Jing Huang

[http://www.chem.umass.edu/people/kaltashovlab/ Kaltashov Lab]

: '''[http://proteopedia.org/wiki/index.php/Chengfeng_Ren Molecular Playground/Inteferon]'''[Revised], Chengfeng Ren
: ''' [[Molecular Playground/Transferrin]]''', Khaja Muneeruddin, Jake Pawlowski
: '''[[Molecular Playground/Velaglucerase]]''', Adriana Kita
: '''[[Molecular Playground/Antithrombin-Heparin]]''', Yunlong Zhao

[http://people.chem.umass.edu/knapplab/?q=knappchem/index.html/ Knapp Lab]
: '''[[Molecular Playground/Prolyl Hydroxylase Domain (PHD) Enzyme]]''', Cristina Martin, Serap Pektas '''Best CBI Molecule Proteopedia Page 2010''' '''***New Fall 2014***'''
: '''[[Molecular Playground/FIH]]''', Vanessa Chaplin, Cornelius Taabazuing, Breanne Holmes, John Hangasky

[http://people.chem.umass.edu/mmaroney/ Maroney Lab]

: '''[[Molecular Playground/UreE]]''', Priyanka Basak, '''New 2014'''
: '''[[Molecular Playground/Nickel Superoxide Dismutase]]''', Carolyn Carr
: '''[[Molecular Playground/CsoR and RcnR]]''', Heidi Hu, Carolyn Carr, Hsin-Ting (Tiffany )Huang, '''Revised 2014'''
: '''[[Molecular Playground/HypA]]''', Heidi Hu

[http://www.chem.umass.edu/~cmartin/ Martin Lab]

: **'''[[Molecular Playground/T7 RNAP Conformations]]''', Luis E. Ramirez-Tapia '''Best CBI Molecule 2011'''
: '''[[Molecular Playground/T7 RNA Polymerase (7 mer int)]]''', Ankit Vahia
: '''[[Molecular Playground/Human mtRNA pol]]''', Ketan Mathavan

[http://www.peytonlab.org/ Peyton Lab]
: '''[[Molecular Playground/IntegrinBeta1]]''', Lauren Jansen, Lauren Barney, Elizabeth Brooks, Alyssa Schwartz ***'''NEW FALL 2014''''***
: '''[[Molecular Playground/MMP14]]''', Lauren Jansen
: '''[[Molecular Playground/C-Raf]]''', Thuy Nguyen
: '''[[Molecular Playground/FAK]]''', Dannielle Ryman

[http://roberts.openwetware.org/ Roberts Lab]

: '''[[Molecular Playground/Taxol]]''', Elizabeth Cummings, Rohan Patil, Sarah Wilson ***'''Revised Fall 2014'''***
: '''[[Molecular Playground/Insulin]]''', Whitney Stoppel

[http://www.umass.edu/rotellogroup/ Rotello Lab]

: '''[[Reverse transcriptase|Molecular Playground/Reverse Transcriptase]]''', Daniel Moyano-Marino
: '''[[Molecular Playground/Pancreatic Lipase]]''', Rui Tang
: '''[[Molecular playground/Chymotrypsin]]''', Brad
: '''[[Molecular Playground/Targeting Peptide]]''', David Solfiell

[http://http://www.umass.edu/schiffman/ Schiffman Lab]
: '''[[Molecular Playground/Streptavidin]]''', Katrina Rieger

[http://www.biochem.umass.edu/faculty/danny-j-schnell Schnell Lab]
: '''[[Molecular Playground/Tic40]]''', Mine Canakci

Tew Lab

: '''[[Molecular Playground/HIV Tat]]''', Brittany deRonde, Coralie Backlund

Thayumanavan Lab
: '''[[Interleukin-1 beta]]''', Huan He
: '''[[Molecular Playground/Lysozyme ]]''', Daniella Gonzalez
: '''[[Molecular Playground/Beta-galactosidase]]''', Judy Ventura
: '''[[User:Krishna Reddy Ragupathi|Molecular Playground/Carbonic Anhydrase]]''', Krishna Reddy Raghupathi
: '''[[User:Rami Rajasekhar Reddy|Molecular Playground/Avidin]]''', Rami Rajasekar Reddy
: '''[[Molecular Playground/Biotin binding avidin]]''', Diego Amado
: '''[[User:Jiaming Zhuang|Molecular Playground/MMP12]]''', Jiaming Zhuang
: ''' [[User:Jing Guo|Molecular Playground/Gluconase]]''', Jing Guo
: '''[[Molecular Playground/Glutathione Reductase]]''', Reuben Chacko

Thayumanavan & Vachet Labs

: '''[[Molecular Playground/Trypsin]]''', Gladys Murage

[http://people.chem.umass.edu/thompson/index.html Thompson Lab]

: '''[[Molecular Playground/Bacterial Chemotaxis Complex]]''', Elizabeth R. Haglin, Maryam Kashefi
: '''[[Molecular Playground/CheA]]''', Elizabeth R. Haglin
: '''[[Molecular Playground/Bacterial Chemotaxis Receptors]]''', Lynmarie K. Thompson, Shiela M. Jones
: '''[[Molecular Playground/Cytoplasmic domain of chemoreceptor of Thermotoga maritima]]''', Xuni Li

[http://www.chem.umass.edu/~vachet/index.html Vachet Lab]

: '''[[Molecular Playground/Human Serum Albumin]]''', Mahalia Serrano ''*Revised 2014*''
: '''[[Molecular playground/beta 2 microglobulin]]''', Nick Borotto, Tyler Marcinko ***'''Revised Fall 2014'''*** '''Best CBI Molecule Jmol scenes 2010'''
: '''[[Molecular Playground/Myoglobin]]''', Yuping Zhou
:'''[[Molecular Playground/ Copper-Zinc Superoxide Dismutase]]''', Shaynah Browne
: '''[[Molecular Playground/Ubiquitin salt bridge discussion]]''', Zhe Zhang

Other Molecules of interest

: '''[[Molecular Playground/PcrA Helicase]]''', Luis E Ramirez-Tapia, [http://www.chem.umass.edu/~cmartin/ Martin Lab]

Other Laboratories

:'''[[Molecular Playground/4'-PHOSPHOPANTETHEINYL TRANSFERASE (Sfp)]]''', Jon Amoroso, Gitanjeli Prasad, Lawrence Sheringham Borketey, Schnarr lab
: '''[[6-deoxyerythronolide_B_synthase_(DEBS)|Molecular Playground/6-Deoxyerythronolide B Synthase]]''', Tsung-Yi Lin, Jon Amoroso, Lawrence Sheringham Borketey, Schnarr lab
: '''[[Molecular Playground/ACP apo]]''', Gitanjeli Prasad, Schnarr lab

: '''[[Molecular Playground/cytoplasmic domain of a serine chemotaxis receptor]]''', Meili Yang, Weis lab

: '''[[Molecular Playground/Alginate-Fall2011]]''', Joe White, David Griffi, Bhatia Lab
: '''[[Molecular Playground/Poly(ethylene glycol)]]''', Erika M. Saffer, Bhatia Lab

: '''[[Molecular Playground/YKL-40]]''', Ralph A. Francescone III, [http://www.bio.umass.edu/mcb/faculty/Shao.html Shao Lab]

: '''[[Molecular Playground/BLG|Molecular Playground/β-lactoglobulin]]''', Daniel Seeman, Dubin Lab

: '''[[Molecular Playground/ADAM13]]''', Genevieve Abbruzzese, Alfandari Lab

==Instructions==

'''The goal is for students to work collaboratively to create one excellent CBI Molecule for each CBI research group, to be featured on the CBI website.'''

Fall 2014: Complete steps 1-3 before the CBI Molecule Workshop on 11/19; bring your computer to the CBI Molecule Workshop in ISB 321. You will be able to start work on your CBI Molecule at the workshop. You should plan to stay and finish your molecule after the workshop (or as soon as possible) while the instructions and ideas are fresh in your mind.

A successful CBI Molecule is an interesting, nontechnical description of a molecule related to your group's research. It should tell an interesting story with few words and many green scenes. Green scenes should be clear and attractive illustrations that tell your story. It's great if you can end with a brief statement about how it relates to your group's research goals.

1. If you don't already have one, request a Proteopedia account and log in.

2. Read through the rest of the instructions, do the tutorials below, and look at any existing CBI Molecules from your research group. Talk with other CBI students in your research group and decide how you will collaborate to make an improved CBI Molecule related to your group's research. Develop ideas for the scenes that each of you will make.

3. Get started working in Proteopedia. Make yourself a sandbox page: Enter "User:Your Name/Sandbox 1" (omit quotes) in the search box, then follow instructions to edit this page. Practice entering text, inserting a structure window with scrolling text (green 4-square button on the right), and creating a green scene. Explore the HELP links below to learn how to make a green scene. You can look at other Proteopedia pages in edit mode to see how different effects (like colored text) are achieved (but this won't show you how the scene effects are made).

'''Complete the above steps before the CBI Molecule Workshop.'''

4. When editing an existing CBI Molecule, leave the original intact and make a copy that you will edit so that it retains the full author list of all authors that contributed. You are encouraged to collaborate on the CBI Molecules, but everyone will need to log in as themself and create a green scene to get credit for their work and to appear as authors on the final list. If you are the sole student from your group enrolled in Chalk Talk and have not previously made a CBI Molecule, try to convince another group member who has previously made a CBI Molecule (see list above) to join us for the workshop so that you can work together.

5. Your final pages should be called "Molecular Playground/your molecule" and should have links on this CBI Molecule page -- put the new ones first on the list from your research group. Label them "in progress" until you are finished, and then "new 2014" or "revised 2014". Minimize text; tell your story with green scenes!

6. When your CBI Molecule is complete, send Lynmarie an email to nominate a cool scene/clever caption you would like to feature at the Molecular Playground (include a link to your CBI Molecule, name of the green scene, and caption).

==Links to HELP pages==
Overview at [[Help:Contents ]]

See also [[Help:Getting_Started_in_Proteopedia]]

Videos showing how to use Proteopedia:
[[Proteopedia:Video_Guide]]
(Sometimes slow -- if you pause the video and wait awhile it will download and then you can play it without interruptions)

[[Proteopedia:DIY:Scenes|Proteopedia Scenes: Do It Yourself]]
gives succinct step by step instructions on how to create a molecular scene.

A powerpoint-like set of slides that walks a user through the process of creating a new page and a new molecular scene/green link.
[http://www.proteopedia.org/wiki/images/1/1b/2009_07_13_Proteopedia_Workshop.pdf Proteopedia Workshop Slides]

[[Proteopedia:Guidelines for Ethical Writing]].
Please pay attention to the section about images. There are links to examples of images re-used with explicit permission.

For other help resources, click on [[Help:Contents|Help]] in the ''navigation'' box at the upper left of every page in Proteopedia.

Brittany deRonde/Sandbox 1

2014-11-21T17:51:22Z

Coralie Backlund:

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground].

== '''Introduction''' ==

'''HIV Tat''', or simply Tat, is a human immunodeficiency virus (HIV) gene that regulates transcription of HIV dsRNA.<ref>Sodroski ''et al. Science.'' '''1985'''. 227, 171-173. [http://www.jstor.org/stable/1695050] http://www.jstor.org/stable/1695050</ref> Tat, which stands for trans-activator of transcription, contains 86 amino acid residues in its sequence.<ref>Arya ''et al. Science'' '''1985'''. 229, 69-73.</ref><ref>Sodroski ''et al. Science.'' '''1985'''. 229, 74-77.</ref> <Structure load='1TIV' size='500' frame='true' align='right' caption='HIV Tat. Grey residues = polar groups, Pink residues= hydrophobic residues, and Blue residues= protein transduction domain' scene='Brittany_deRonde/Sandbox_1/Hiv_tat/2' /> It is released by HIV infected cells in order to enhance replication of the virus.<ref>Dayton ''et al. Cell.'' '''1986'''. 44, 941-947.</ref><ref>Fisher ''et al. Nature.'' '''1986'''. 320, 367-371.</ref> Nanomolar concentrations of Tat have been reported in the blood of HIV-1 infected people.<ref>Xiao ''et al. Proc. Natl. Acad. Sci. USA.'' '''2000'''. 97, 11466-11471.</ref> When the protein enters non-infected cells, the transcription efficiency increases. It is estimated that transcription levels are 10 to 100 fold higher during the transcription elongation stage when compared to normal basal transcription.<ref>Ensoli ''et al. Nature.'' '''1990'''. 345, 84-86.</ref> Green and Lowenstein, and Frankel and Pabo independently published studies in 1988 that demonstrated that Tat had the ability to cross cellular membranes and initiate transcription of HIV dsRNA.<ref>Green ''et al. Cell.'' '''1988'''. 55, 1179-1188.</ref><ref>Frankel ''et al. Cell.'' '''1988'''. 55, 1189-1193.</ref> These were the first known reports of a '''cell penetrating peptide (CPP)'''.

== '''Cell Penetrating Peptides''' ==

'''Cell penetrating peptides (CPPs)''' are proteins with the ability to cross cellular membranes and facilitate the uptake of various cargo, such as small molecules, siRNA, and small DNA fragments.<ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref> Such cargo can be associated via covalent or non-covalent interactions. Tat is considered a CPP because it contains a protein transduction domain (PTD). PTDs are cation-rich sequences of 10-30 residues, usually containing several Lysine and/or Arginine residues.<ref>Wender ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 452-472.</ref>

[[Image:Arginine1.jpg]] [[Image:Lysine1.jpg]]

These sequences improve interactions between CPPs and cellular membranes and can help them enter cells. The (amino acid <scene name='Brittany_deRonde/Sandbox_1/Hiv_tat/3'>residues 47-57</scene>) of HIV-1 Tat comprise the protein transduction region of this protein. This arginine rich, 11 amino-acid sequence, <scene name='47/475184/Tat_arg/3'>YGRKKRRQRRR</scene> is now referred to as the TAT peptide, and has be shown to have improved cellular uptake compared to Tat.

Since the discovery of HIV Tat, other Arginine-rich, natural CPPs have been discovered including, penetratin from Drosophila antennapedia protein, sweet arrow peptide (SAP), HIV-1 Rev, flock house virus (FHV) coat, brome mosaic virus (BMV) Gag, human T-cell lymphotrophic virus (HTLV)-II Rex, and the nuclear localization signal (NLS) from nucleoplasmin. <ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref><ref>Prochiantz ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 448-451.</ref> The PTD sequences for these proteins can be found in the table below.

[[Image:Arginine_Rich_Peptides.png]]

== '''Tat-mediated Transduction''' ==

The PTD in HIV Tat is highly cationic. When charged residues (Arginine or Lysine) are replaced with neutral residues (Alanine), cellular uptake decreases. It is likely that the cationic charge is necessary for electrostatic interactions with components of the cellular membrane, such as lipid head groups and proteoglycans.<ref>Ziegler ''et al. Biochemistry'' '''2003'''. 42, 9185-9194.</ref><ref>Goncalves ''et al. Biochemistry.'' '''2005'''. 44, 2692-2702.</ref> Other interactions may also play a role in interactions with the cellular membrane, such as hydrophobic and non-covalent interactions.<ref>Ziegler ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 580-597.</ref> Although cellular uptake is not directly dependent upon architecture (branched vs. linear), it is dependent upon the number of Arginine residues. Polymers with less than five arginine residues are unable to enter cells.<ref>Wender ''et al. Proc. Natl. Acad. Sci.'' '''2000'''. 97, 13003-13008.</ref> As the number of residues increases, cellular uptake improves up until 15 residues where polymers can enter cells but with hindered efficiencies.<ref>Mitchell ''et al. J. Pept. Res.'' '''2000'''. 56, 318-325.</ref><ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref> The exact mechanism of cellular uptake is still highly debated in the literature, but a couple of the proposed methods are discussed below.

==Transduction==
Early studies indicated that HIV Tat primarily entered cells through transduction, meaning that the protein directly crossed the cell membranes. However, for most of these studies, cells were fixed with formaldehyde prior to sample analysis. In 2003, it was discovered that by fixing cells prior to analysis, it permeablized the cell membranes and resulted in artificially high cellular uptake values. Fixed cells showed higher concentrations of Tat in nuclei compared to non-fixed cells where it was localized in the cytoplasm. <ref>Richard ''et al. J. Biol. Chem.'' '''2003'''. 278, 585-590.</ref> Studies also showed that FACS could not tell the difference between internalized cells and those proteins that were only bound to the cellular membranes. Cellular uptake studies performed at 4°C and under conditions of ATP depletion show that a small percentage of CPPs can enter cells and also suggests that a majority of the protein enters cells through energy dependent pathways.<ref>Drin ''et al. J. Biol. Chem.'' '''2003'''. 276, 31192-31201.</ref> Some groups have also studied transduction of Tat using model vesicles. <ref>Ziegler ''et al. Biochemistry.'' '''2003'''. 278, 9185-9194.</ref>

==Endocytosis==
Another proposed mechanism for cellular uptake is endocytosis, specifically macropinocytosis. This form of fluid phase endocytosis is less known that other types (phagocytosis, clathrin, and coated pit), it is believed to occur in all cell types and enable the uptake of large molecules. <ref>Gump ''et al. Trends in Molecular Medicine.'' '''2007'''. 13, 443-448.</ref> During this process, actin protrusions fold around molecules and the surrounding medium and enable cellular uptake.

== '''My Research Interests''' ==
As part of the [http://www.pse.umass.edu/gtew/index.html Tew Research Group] in the [http://www.pse.umass.edu Department of Polymer Science and Engineering] at the University of Massachusetts, Amherst, I am interested in the design of Tat-inspired CPPs for drug and gene delivery. Using functionalized oxanorbornene derivatives as monomers (as shown below), ring opening metathesis polymerization (ROMP) is performed using Grubbs' 3rd generation catalyst to synthesize cationic CPP mimics. Hydrophobicity, charge, aromaticity, and pi-electronics of the monomer structures can be optimized to achieve better cellular uptake and cell viability by changing R1 and R2.

[[Image:norbornenederivatives.png]]

== '''Acknowledgments''' ==
The Tew research group is gratefully acknowledged for their contributions to the design and content of this page.

== '''References''' ==
{{Reflist}}

Brittany deRonde/Sandbox 1

2014-11-21T17:47:36Z

Coralie Backlund:

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground].

== '''Introduction''' ==

'''HIV Tat''', or simply Tat, is a human immunodeficiency virus (HIV) gene that regulates transcription of HIV dsRNA.<ref>Sodroski ''et al. Science.'' '''1985'''. 227, 171-173. [http://www.jstor.org/stable/1695050] http://www.jstor.org/stable/1695050</ref> Tat, which stands for trans-activator of transcription, contains 86 amino acid residues in its sequence.<ref>Arya ''et al. Science'' '''1985'''. 229, 69-73.</ref><ref>Sodroski ''et al. Science.'' '''1985'''. 229, 74-77.</ref> <Structure load='1TIV' size='500' frame='true' align='right' caption='HIV Tat. Grey residues = polar groups, Pink residues= hydrophobic residues, and Blue residues= protein transduction domain' scene='Brittany_deRonde/Sandbox_1/Hiv_tat/2' /> It is released by HIV infected cells in order to enhance replication of the virus.<ref>Dayton ''et al. Cell.'' '''1986'''. 44, 941-947.</ref><ref>Fisher ''et al. Nature.'' '''1986'''. 320, 367-371.</ref> Nanomolar concentrations of Tat have been reported in the blood of HIV-1 infected people.<ref>Xiao ''et al. Proc. Natl. Acad. Sci. USA.'' '''2000'''. 97, 11466-11471.</ref> When the protein enters non-infected cells, the transcription efficiency increases. It is estimated that transcription levels are 10 to 100 fold higher during the transcription elongation stage when compared to normal basal transcription.<ref>Ensoli ''et al. Nature.'' '''1990'''. 345, 84-86.</ref> Green and Lowenstein, and Frankel and Pabo independently published studies in 1988 that demonstrated that Tat had the ability to cross cellular membranes and initiate transcription of HIV dsRNA.<ref>Green ''et al. Cell.'' '''1988'''. 55, 1179-1188.</ref><ref>Frankel ''et al. Cell.'' '''1988'''. 55, 1189-1193.</ref> These were the first known reports of a '''cell penetrating peptide (CPP)'''.

== '''Cell Penetrating Peptides''' ==

'''Cell penetrating peptides (CPPs)''' are proteins with the ability to cross cellular membranes and facilitate the uptake of various cargo, such as small molecules, siRNA, and small DNA fragments.<ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref> Such cargo can be associated via covalent or non-covalent interactions. Tat is considered a CPP because it contains a protein transduction domain (PTD). PTDs are cation-rich sequences of 10-30 residues, usually containing several Lysine and/or Arginine residues.<ref>Wender ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 452-472.</ref>

[[Image:Arginine1.jpg]] [[Image:Lysine1.jpg]]

These sequences improve interactions between CPPs and cellular membranes and can help them enter cells. The (amino acid <scene name='Brittany_deRonde/Sandbox_1/Hiv_tat/3'>residues 47-57</scene>) of HIV-1 Tat comprise the protein transduction region of this protein. This arginine rich, 11 amino-acid sequence, <scene name='47/475184/Tat_arg/1'>YGRKKRRQRRR</scene> is now referred to as the TAT peptide, and has be shown to have improved cellular uptake compared to Tat.

Since the discovery of HIV Tat, other Arginine-rich, natural CPPs have been discovered including, penetratin from Drosophila antennapedia protein, sweet arrow peptide (SAP), HIV-1 Rev, flock house virus (FHV) coat, brome mosaic virus (BMV) Gag, human T-cell lymphotrophic virus (HTLV)-II Rex, and the nuclear localization signal (NLS) from nucleoplasmin. <ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref><ref>Prochiantz ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 448-451.</ref> The PTD sequences for these proteins can be found in the table below.

[[Image:Arginine_Rich_Peptides.png]]

== '''Tat-mediated Transduction''' ==

The PTD in HIV Tat is highly cationic. When charged residues (Arginine or Lysine) are replaced with neutral residues (Alanine), cellular uptake decreases. It is likely that the cationic charge is necessary for electrostatic interactions with components of the cellular membrane, such as lipid head groups and proteoglycans.<ref>Ziegler ''et al. Biochemistry'' '''2003'''. 42, 9185-9194.</ref><ref>Goncalves ''et al. Biochemistry.'' '''2005'''. 44, 2692-2702.</ref> Other interactions may also play a role in interactions with the cellular membrane, such as hydrophobic and non-covalent interactions.<ref>Ziegler ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 580-597.</ref> Although cellular uptake is not directly dependent upon architecture (branched vs. linear), it is dependent upon the number of Arginine residues. Polymers with less than five arginine residues are unable to enter cells.<ref>Wender ''et al. Proc. Natl. Acad. Sci.'' '''2000'''. 97, 13003-13008.</ref> As the number of residues increases, cellular uptake improves up until 15 residues where polymers can enter cells but with hindered efficiencies.<ref>Mitchell ''et al. J. Pept. Res.'' '''2000'''. 56, 318-325.</ref><ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref> The exact mechanism of cellular uptake is still highly debated in the literature, but a couple of the proposed methods are discussed below.

==Transduction==
Early studies indicated that HIV Tat primarily entered cells through transduction, meaning that the protein directly crossed the cell membranes. However, for most of these studies, cells were fixed with formaldehyde prior to sample analysis. In 2003, it was discovered that by fixing cells prior to analysis, it permeablized the cell membranes and resulted in artificially high cellular uptake values. Fixed cells showed higher concentrations of Tat in nuclei compared to non-fixed cells where it was localized in the cytoplasm. <ref>Richard ''et al. J. Biol. Chem.'' '''2003'''. 278, 585-590.</ref> Studies also showed that FACS could not tell the difference between internalized cells and those proteins that were only bound to the cellular membranes. Cellular uptake studies performed at 4°C and under conditions of ATP depletion show that a small percentage of CPPs can enter cells and also suggests that a majority of the protein enters cells through energy dependent pathways.<ref>Drin ''et al. J. Biol. Chem.'' '''2003'''. 276, 31192-31201.</ref> Some groups have also studied transduction of Tat using model vesicles. <ref>Ziegler ''et al. Biochemistry.'' '''2003'''. 278, 9185-9194.</ref>

==Endocytosis==
Another proposed mechanism for cellular uptake is endocytosis, specifically macropinocytosis. This form of fluid phase endocytosis is less known that other types (phagocytosis, clathrin, and coated pit), it is believed to occur in all cell types and enable the uptake of large molecules. <ref>Gump ''et al. Trends in Molecular Medicine.'' '''2007'''. 13, 443-448.</ref> During this process, actin protrusions fold around molecules and the surrounding medium and enable cellular uptake.

== '''My Research Interests''' ==
As part of the [http://www.pse.umass.edu/gtew/index.html Tew Research Group] in the [http://www.pse.umass.edu Department of Polymer Science and Engineering] at the University of Massachusetts, Amherst, I am interested in the design of Tat-inspired CPPs for drug and gene delivery. Using functionalized oxanorbornene derivatives as monomers (as shown below), ring opening metathesis polymerization (ROMP) is performed using Grubbs' 3rd generation catalyst to synthesize cationic CPP mimics. Hydrophobicity, charge, aromaticity, and pi-electronics of the monomer structures can be optimized to achieve better cellular uptake and cell viability by changing R1 and R2.

[[Image:norbornenederivatives.png]]

== '''Acknowledgments''' ==
The Tew research group is gratefully acknowledged for their contributions to the design and content of this page.

== '''References''' ==
{{Reflist}}

Coralie Backlund/Sandbox

2014-11-19T18:06:33Z

Coralie Backlund:

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground].

== '''Introduction''' ==

'''HIV Tat''', or simply Tat, is a human immunodeficiency virus (HIV) gene that regulates transcription of HIV dsRNA.<ref>Sodroski ''et al. Science.'' '''1985'''. 227, 171-173. [http://www.jstor.org/stable/1695050] http://www.jstor.org/stable/1695050</ref> Tat, which stands for trans-activator of transcription, contains 86 amino acid residues in its sequence.<ref>Arya ''et al. Science'' '''1985'''. 229, 69-73.</ref><ref>Sodroski ''et al. Science.'' '''1985'''. 229, 74-77.</ref> <Structure load='1TIV' size='500' frame='true' align='right' caption='HIV Tat. Grey residues = polar groups, Pink residues= hydrophobic residues, and Blue residues= protein transduction domain' scene='Brittany_deRonde/Sandbox_1/Hiv_tat/2' /> It is released by HIV infected cells in order to enhance replication of the virus.<ref>Dayton ''et al. Cell.'' '''1986'''. 44, 941-947.</ref><ref>Fisher ''et al. Nature.'' '''1986'''. 320, 367-371.</ref> Nanomolar concentrations of Tat have been reported in the blood of HIV-1 infected people.<ref>Xiao ''et al. Proc. Natl. Acad. Sci. USA.'' '''2000'''. 97, 11466-11471.</ref> When the protein enters non-infected cells, the transcription efficiency increases. It is estimated that transcription levels are 10 to 100 fold higher during the transcription elongation stage when compared to normal basal transcription.<ref>Ensoli ''et al. Nature.'' '''1990'''. 345, 84-86.</ref> Green and Lowenstein, and Frankel and Pabo independently published studies in 1988 that demonstrated that Tat had the ability to cross cellular membranes and initiate transcription of HIV dsRNA.<ref>Green ''et al. Cell.'' '''1988'''. 55, 1179-1188.</ref><ref>Frankel ''et al. Cell.'' '''1988'''. 55, 1189-1193.</ref> These were the first known reports of a '''cell penetrating peptide (CPP)'''.

== '''Cell Penetrating Peptides''' ==

'''Cell penetrating peptides (CPPs)''' are proteins with the ability to cross cellular membranes and facilitate the uptake of various cargo, such as small molecules, siRNA, and small DNA fragments.<ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref> Such cargo can be associated via covalent or non-covalent interactions. Tat is considered a CPP because it contains a protein transduction domain (PTD). PTDs are cation-rich sequences of 10-30 residues, usually containing several Lysine and/or Arginine residues.<ref>Wender ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 452-472.</ref>

[[Image:Arginine1.jpg]] [[Image:Lysine1.jpg]]

These sequences improve interactions between CPPs and cellular membranes and can help them enter cells. The PTD sequence in Tat is YGRKKRRQRRR (amino acid <scene name='Brittany_deRonde/Sandbox_1/Hiv_tat/3'>residues 47-57</scene>), which is Arginine-rich. This (<scene name='60/609789/Arginine_rich/2'>TextToBeDisplayed</scene>11 amino-acid sequence) is now referred to as the TAT peptide, and has be shown to have improved cellular uptake compared to Tat.

Since the discovery of HIV Tat, other Arginine-rich, natural CPPs have been discovered including, penetratin from Drosophila antennapedia protein, sweet arrow peptide (SAP), HIV-1 Rev, flock house virus (FHV) coat, brome mosaic virus (BMV) Gag, human T-cell lymphotrophic virus (HTLV)-II Rex, and the nuclear localization signal (NLS) from nucleoplasmin. <ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref><ref>Prochiantz ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 448-451.</ref> The PTD sequences for these proteins can be found in the table below.

[[Image:Arginine_Rich_Peptides.png]]

== '''Tat-mediated Transduction''' ==

The PTD in HIV Tat is highly cationic. When charged residues (Arginine or Lysine) are replaced with neutral residues (Alanine), cellular uptake decreases. It is likely that the cationic charge is necessary for electrostatic interactions with components of the cellular membrane, such as lipid head groups and proteoglycans.<ref>Ziegler ''et al. Biochemistry'' '''2003'''. 42, 9185-9194.</ref><ref>Goncalves ''et al. Biochemistry.'' '''2005'''. 44, 2692-2702.</ref> Other interactions may also play a role in interactions with the cellular membrane, such as hydrophobic and non-covalent interactions.<ref>Ziegler ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 580-597.</ref> Although cellular uptake is not directly dependent upon architecture (branched vs. linear), it is dependent upon the number of Arginine residues. Polymers with less than five arginine residues are unable to enter cells.<ref>Wender ''et al. Proc. Natl. Acad. Sci.'' '''2000'''. 97, 13003-13008.</ref> As the number of residues increases, cellular uptake improves up until 15 residues where polymers can enter cells but with hindered efficiencies.<ref>Mitchell ''et al. J. Pept. Res.'' '''2000'''. 56, 318-325.</ref><ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref> The exact mechanism of cellular uptake is still highly debated in the literature, but a couple of the proposed methods are discussed below.

==Transduction==
Early studies indicated that HIV Tat primarily entered cells through transduction, meaning that the protein directly crossed the cell membranes. However, for most of these studies, cells were fixed with formaldehyde prior to sample analysis. In 2003, it was discovered that by fixing cells prior to analysis, it permeablized the cell membranes and resulted in artificially high cellular uptake values. Fixed cells showed higher concentrations of Tat in nuclei compared to non-fixed cells where it was localized in the cytoplasm. <ref>Richard ''et al. J. Biol. Chem.'' '''2003'''. 278, 585-590.</ref> Studies also showed that FACS could not tell the difference between internalized cells and those proteins that were only bound to the cellular membranes. Cellular uptake studies performed at 4°C and under conditions of ATP depletion show that a small percentage of CPPs can enter cells and also suggests that a majority of the protein enters cells through energy dependent pathways.<ref>Drin ''et al. J. Biol. Chem.'' '''2003'''. 276, 31192-31201.</ref> Some groups have also studied transduction of Tat using model vesicles. <ref>Ziegler ''et al. Biochemistry.'' '''2003'''. 278, 9185-9194.</ref>

==Endocytosis==
Another proposed mechanism for cellular uptake is endocytosis, specifically macropinocytosis. This form of fluid phase endocytosis is less known that other types (phagocytosis, clathrin, and coated pit), it is believed to occur in all cell types and enable the uptake of large molecules. <ref>Gump ''et al. Trends in Molecular Medicine.'' '''2007'''. 13, 443-448.</ref> During this process, actin protrusions fold around molecules and the surrounding medium and enable cellular uptake.

== '''My Research Interests''' ==
As part of the [http://www.pse.umass.edu/gtew/index.html Tew Research Group] in the [http://www.pse.umass.edu Department of Polymer Science and Engineering] at the University of Massachusetts, Amherst, I am interested in the design of Tat-inspired CPPs for drug and gene delivery. Using functionalized oxanorbornene derivatives as monomers (as shown below), ring opening metathesis polymerization (ROMP) is performed using Grubbs' 3rd generation catalyst to synthesize cationic CPP mimics. Hydrophobicity, charge, aromaticity, and pi-electronics of the monomer structures can be optimized to achieve better cellular uptake and cell viability by changing R1 and R2.

[[Image:norbornenederivatives.png]]

== '''Acknowledgments''' ==
The Tew research group is gratefully acknowledged for their contributions to the design and content of this page.

== '''References''' ==
{{Reflist}}

Coralie Backlund/Sandbox

2014-11-19T17:47:37Z

Coralie Backlund:

One of the [[CBI Molecules]] being studied in the [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground].

== '''Introduction''' ==

'''HIV Tat''', or simply Tat, is a human immunodeficiency virus (HIV) gene that regulates transcription of HIV dsRNA.<ref>Sodroski ''et al. Science.'' '''1985'''. 227, 171-173. [http://www.jstor.org/stable/1695050] http://www.jstor.org/stable/1695050</ref> Tat, which stands for trans-activator of transcription, contains 86 amino acid residues in its sequence.<ref>Arya ''et al. Science'' '''1985'''. 229, 69-73.</ref><ref>Sodroski ''et al. Science.'' '''1985'''. 229, 74-77.</ref> <Structure load='1TIV' size='500' frame='true' align='right' caption='HIV Tat. Grey residues = polar groups, Pink residues= hydrophobic residues, and Blue residues= protein transduction domain' scene='Brittany_deRonde/Sandbox_1/Hiv_tat/2' /> It is released by HIV infected cells in order to enhance replication of the virus.<ref>Dayton ''et al. Cell.'' '''1986'''. 44, 941-947.</ref><ref>Fisher ''et al. Nature.'' '''1986'''. 320, 367-371.</ref> Nanomolar concentrations of Tat have been reported in the blood of HIV-1 infected people.<ref>Xiao ''et al. Proc. Natl. Acad. Sci. USA.'' '''2000'''. 97, 11466-11471.</ref> When the protein enters non-infected cells, the transcription efficiency increases. It is estimated that transcription levels are 10 to 100 fold higher during the transcription elongation stage when compared to normal basal transcription.<ref>Ensoli ''et al. Nature.'' '''1990'''. 345, 84-86.</ref> Green and Lowenstein, and Frankel and Pabo independently published studies in 1988 that demonstrated that Tat had the ability to cross cellular membranes and initiate transcription of HIV dsRNA.<ref>Green ''et al. Cell.'' '''1988'''. 55, 1179-1188.</ref><ref>Frankel ''et al. Cell.'' '''1988'''. 55, 1189-1193.</ref> These were the first known reports of a '''cell penetrating peptide (CPP)'''.

== '''Cell Penetrating Peptides''' ==

'''Cell penetrating peptides (CPPs)''' are proteins with the ability to cross cellular membranes and facilitate the uptake of various cargo, such as small molecules, siRNA, and small DNA fragments.<ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref> Such cargo can be associated via covalent or non-covalent interactions. Tat is considered a CPP because it contains a protein transduction domain (PTD). PTDs are cation-rich sequences of 10-30 residues, usually containing several Lysine and/or Arginine residues.<ref>Wender ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 452-472.</ref>

[[Image:Arginine1.jpg]] [[Image:Lysine1.jpg]]

These sequences improve interactions between CPPs and cellular membranes and can help them enter cells. The PTD sequence in Tat is YGRKKRRQRRR (amino acid <scene name='Brittany_deRonde/Sandbox_1/Hiv_tat/3'>residues 47-57</scene>), which is Arginine-rich. This 11 amino-acid sequence is now referred to as the TAT peptide, and has be shown to have improved cellular uptake compared to Tat.

Since the discovery of HIV Tat, other Arginine-rich, natural CPPs have been discovered including, penetratin from Drosophila antennapedia protein, sweet arrow peptide (SAP), HIV-1 Rev, flock house virus (FHV) coat, brome mosaic virus (BMV) Gag, human T-cell lymphotrophic virus (HTLV)-II Rex, and the nuclear localization signal (NLS) from nucleoplasmin. <ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref><ref>Prochiantz ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 448-451.</ref> The PTD sequences for these proteins can be found in the table below.

[[Image:Arginine_Rich_Peptides.png]]

== '''Tat-mediated Transduction''' ==

The PTD in HIV Tat is highly cationic. When charged residues (Arginine or Lysine) are replaced with neutral residues (Alanine), cellular uptake decreases. It is likely that the cationic charge is necessary for electrostatic interactions with components of the cellular membrane, such as lipid head groups and proteoglycans.<ref>Ziegler ''et al. Biochemistry'' '''2003'''. 42, 9185-9194.</ref><ref>Goncalves ''et al. Biochemistry.'' '''2005'''. 44, 2692-2702.</ref> Other interactions may also play a role in interactions with the cellular membrane, such as hydrophobic and non-covalent interactions.<ref>Ziegler ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 580-597.</ref> Although cellular uptake is not directly dependent upon architecture (branched vs. linear), it is dependent upon the number of Arginine residues. Polymers with less than five arginine residues are unable to enter cells.<ref>Wender ''et al. Proc. Natl. Acad. Sci.'' '''2000'''. 97, 13003-13008.</ref> As the number of residues increases, cellular uptake improves up until 15 residues where polymers can enter cells but with hindered efficiencies.<ref>Mitchell ''et al. J. Pept. Res.'' '''2000'''. 56, 318-325.</ref><ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref> The exact mechanism of cellular uptake is still highly debated in the literature, but a couple of the proposed methods are discussed below.

==Transduction==
Early studies indicated that HIV Tat primarily entered cells through transduction, meaning that the protein directly crossed the cell membranes. However, for most of these studies, cells were fixed with formaldehyde prior to sample analysis. In 2003, it was discovered that by fixing cells prior to analysis, it permeablized the cell membranes and resulted in artificially high cellular uptake values. Fixed cells showed higher concentrations of Tat in nuclei compared to non-fixed cells where it was localized in the cytoplasm. <ref>Richard ''et al. J. Biol. Chem.'' '''2003'''. 278, 585-590.</ref> Studies also showed that FACS could not tell the difference between internalized cells and those proteins that were only bound to the cellular membranes. Cellular uptake studies performed at 4°C and under conditions of ATP depletion show that a small percentage of CPPs can enter cells and also suggests that a majority of the protein enters cells through energy dependent pathways.<ref>Drin ''et al. J. Biol. Chem.'' '''2003'''. 276, 31192-31201.</ref> Some groups have also studied transduction of Tat using model vesicles. <ref>Ziegler ''et al. Biochemistry.'' '''2003'''. 278, 9185-9194.</ref>

==Endocytosis==
Another proposed mechanism for cellular uptake is endocytosis, specifically macropinocytosis. This form of fluid phase endocytosis is less known that other types (phagocytosis, clathrin, and coated pit), it is believed to occur in all cell types and enable the uptake of large molecules. <ref>Gump ''et al. Trends in Molecular Medicine.'' '''2007'''. 13, 443-448.</ref> During this process, actin protrusions fold around molecules and the surrounding medium and enable cellular uptake.

== '''My Research Interests''' ==
As part of the [http://www.pse.umass.edu/gtew/index.html Tew Research Group] in the [http://www.pse.umass.edu Department of Polymer Science and Engineering] at the University of Massachusetts, Amherst, I am interested in the design of Tat-inspired CPPs for drug and gene delivery. Using functionalized oxanorbornene derivatives as monomers (as shown below), ring opening metathesis polymerization (ROMP) is performed using Grubbs' 3rd generation catalyst to synthesize cationic CPP mimics. Hydrophobicity, charge, aromaticity, and pi-electronics of the monomer structures can be optimized to achieve better cellular uptake and cell viability by changing R1 and R2.

[[Image:norbornenederivatives.png]]

== '''Acknowledgments''' ==
The Tew research group is gratefully acknowledged for their contributions to the design and content of this page.

== '''References''' ==
{{Reflist}}

Coralie Backlund/Sandbox

2014-11-19T17:27:32Z

Coralie Backlund: New page: ==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 you...

==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 '''Coralie Backlund/Sandbox'''. 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 ==

== Disease ==

== 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/>