Biochemistry; Lipid Bilayers; Membranes; Molecular Biology; Protein Folding
Our work is in two areas: interactions of transmembrane helices, and insertion of peptides into membranes. We have discovered a peptide that is soluble in water but can insert itself across a membrane if the local pH is low. This peptide can be used to deliver molecules into cells, driving delivery with low pH. Phalloidin, dyes, and PNAs can be delivered, and research is aimed at finding the rules for transport. We have also found that the peptide can target tumors when injected into mice, and we are pursuing this exciting finding with imaging and therapy in mind. Our studies of helix interactions are now concerned with their roles in viral envelope proteins and single TM receptors, and the use of druglike molecules to modulate the activity of receptors or to attenuate virulence by binding to the TM regions.
Specialized Terms: Helix Interactions; Lipid Bilayers; Membranes; Physical Biochemistry; Protein Folding; Proteins
Extensive Research Description
Folding and Oligomerization of Membrane Proteins
We are interested in how the primary sequences of membrane proteins determine their three dimensional structures, and hence their functions. The folding of integral membrane proteins clearly differs from that of soluble proteins since the membrane environment imposes constraints on polypeptide secondary and tertiary structural features quite different from those imposed by an aqueous environment.
A conceptual underpinning for much of the work in the group is that, for helical transmembrane proteins, the protein folding process can be considered to occur in two kinetically separated and therefore energetically distinct stages. First, transbilayer alpha-helices are formed (stage I) and second, the helices interact within the bilayer to form a specific globular tertiary structure (stage II).
In the case of oligomerization events, monomeric proteins are synthesized and inserted into the membrane, and these monomers subsequently interact in a side-to-side fashion to form complexes that involve helix-helix interactions similar to those found within polytopic helical membrane proteins.
Our most recent work in this area is to examine the association of transmembrane domains (TMs) involved in signaling by receptors that have a single TM, where the signaling mechanism is mediated by TM interactions (see essay below).
Uses and mechanism of pH dependant Tm insertion (in collaboration with the Reshetnyak/Andreev lab, University of Rhode Island)
We have previously observed the spontaneous, pH-dependent insertion of a water-soluble peptide to form a helix across lipid bilayers. We have now used a related peptide, pH (low) insertion peptide (pHLIP), to translocate cargo molecules attached to its C terminus across the plasma membranes of living cells. Translocation is selective for low pH, and various types of cargo molecules attached by disulfides can be released by reduction in the cytoplasm, including peptide nucleic acids, a cyclic peptide (phalloidin), and organic compounds. Because a high extracellular acidity is characteristic of a variety of pathological conditions (such as tumors, infarcts, stroke-afflicted tissue, atherosclerotic lesions, sites of inflammation or infection, or damaged tissue resulting from trauma), the pH (low) insertion peptide may prove a useful tool for selective delivery of agents for drug therapy, diagnostic imaging, genetic control, or cell regulation.
We have recently shown that pHLIP can localize and map acidic foci in kidneys, tumors and inflammatory sites in vivo. In a mouse breast adenocarcinoma model, fluorescently labeled pHLIP finds solid acidic tumors with high accuracy and accumulates in them even at a very early stage of tumor development. The peptide has three states: soluble in water, bound to the surface of a membrane, and inserted across the membrane as an alpha-helix. At physiological pH, the equilibrium is toward water, which explains its low affinity for cells in healthy tissue; at acidic pH, titration of Asp residues shifts the equilibrium toward membrane insertion and tissue accumulation. The pHLIP technology introduces a new concept to detect, target, and possibly treat acidic diseased tissue by employing the selective insertion and folding of membrane peptides.
We are continuing our work on the fundamental mechanism and capabilities of the pHLIP technology.
Mechanism and uses of a peptide that targets tumors and other acidic tissues in vivo
Andreev, O.A., et al. (2007). Mechanism and uses of a peptide that targets tumors and other acidic tissues in vivo. Proc. Natl. Acad. Sci. (USA) 104:7893-8.
A monomeric membrane peptide that lives in three worlds: in solution, attached to and inserted across lipid bilayers
Reshetnyak, Y. K., Segala, M., Andreev, O. A. and Engelman, D. M. "A monomeric membrane peptide that lives in three worlds: in solution, attached to and inserted across lipid bilayers" Biophys. J. (2007) In press
Dynamic helix interactions in transmembrane signaling
Matthews, E.E., Zoonens, M., and Engelman, D.M. (2006). Dynamic helix interactions in transmembrane signaling. Cell 127:447-50.
Translocation of molecules into cells by pH-dependent insertion of a transmembrane helix
Reshetnyak, Y. K., Andreev, O. A., Lehnert, U. and Engelman, D. M. "Translocation of molecules into cells by pH-dependent insertion of a transmembrane helix" PNAS 103, 6460-6465 (2006)
Membranes are more mosaic than fluid
Engelman, D. M. "Membranes are more mosaic than fluid" Nature 438, 578-80 (2005)
Full List of PubMed Publications
- Wyatt LC, Moshnikova A, Crawford T, Engelman DM, Andreev OA, Reshetnyak YK: Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors. Proc Natl Acad Sci U S A. 2018 Mar 20; 2018 Mar 5. PMID: 29507241
- Karabadzhak AG, Petti LM, Barrera FN, Edwards APB, Moya-Rodríguez A, Polikanov YS, Freites JA, Tobias DJ, Engelman DM, DiMaio D: Two transmembrane dimers of the bovine papillomavirus E5 oncoprotein clamp the PDGF β receptor in an active dimeric conformation. Proc Natl Acad Sci U S A. 2017 Aug 29; 2017 Aug 14. PMID: 28808001
- Wyatt LC, Lewis JS, Andreev OA, Reshetnyak YK, Engelman DM: Applications of pHLIP Technology for Cancer Imaging and Therapy. Trends Biotechnol. 2017 Jul; 2017 Apr 21. PMID: 28438340
- Golijanin J, Amin A, Moshnikova A, Brito JM, Tran TY, Adochite RC, Andreev GO, Crawford T, Engelman DM, Andreev OA, Reshetnyak YK, Golijanin D: Targeted imaging of urothelium carcinoma in human bladders by an ICG pHLIP peptide ex vivo. Proc Natl Acad Sci U S A. 2016 Oct 18; 2016 Sep 29. PMID: 27688767
- Anderson M, Moshnikova A, Engelman DM, Reshetnyak YK, Andreev OA: Probe for the measurement of cell surface pH in vivo and ex vivo. Proc Natl Acad Sci U S A. 2016 Jul 19; 2016 Jul 5. PMID: 27382181
- Svoronos AA, Engelman DM, Slack FJ: OncomiR or Tumor Suppressor? The Duplicity of MicroRNAs in Cancer. Cancer Res. 2016 Jul 1; 2016 Jun 20. PMID: 27325641
- Popot JL, Engelman DM: Membranes Do Not Tell Proteins How To Fold. Biochemistry. 2016 Jan 12; 2015 Dec 19. PMID: 26649989
- Heim EN, Marston JL, Federman RS, Edwards AP, Karabadzhak AG, Petti LM, Engelman DM, DiMaio D: Biologically active LIL proteins built with minimal chemical diversity. Proc Natl Acad Sci U S A. 2015 Aug 25; 2015 Aug 10. PMID: 26261320
- Tapmeier TT, Moshnikova A, Beech J, Allen D, Kinchesh P, Smart S, Harris A, McIntyre A, Engelman DM, Andreev OA, Reshetnyak YK, Muschel RJ: The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions. Proc Natl Acad Sci U S A. 2015 Aug 4; 2015 Jul 20. PMID: 26195776
- Cheng CJ, Bahal R, Babar IA, Pincus Z, Barrera F, Liu C, Svoronos A, Braddock DT, Glazer PM, Engelman DM, Saltzman WM, Slack FJ: MicroRNA silencing for cancer therapy targeted to the tumour microenvironment. Nature. 2015 Feb 5; 2014 Nov 17. PMID: 25409146
- Deacon JC, Engelman DM, Barrera FN: Targeting acidity in diseased tissues: mechanism and applications of the membrane-inserting peptide, pHLIP. Arch Biochem Biophys. 2015 Jan 1; 2014 Nov 18. PMID: 25444855
- Karabadzhak AG, An M, Yao L, Langenbacher R, Moshnikova A, Adochite RC, Andreev OA, Reshetnyak YK, Engelman DM: pHLIP-FIRE, a cell insertion-triggered fluorescent probe for imaging tumors demonstrates targeted cargo delivery in vivo. ACS Chem Biol. 2014 Nov 21; 2014 Sep 10. PMID: 25184440
- Viola-Villegas NT, Carlin SD, Ackerstaff E, Sevak KK, Divilov V, Serganova I, Kruchevsky N, Anderson M, Blasberg RG, Andreev OA, Engelman DM, Koutcher JA, Reshetnyak YK, Lewis JS: Understanding the pharmacological properties of a metabolic PET tracer in prostate cancer. Proc Natl Acad Sci U S A. 2014 May 20; 2014 May 1. PMID: 24785505
- Cohen EB, Jun SJ, Bears Z, Barrera FN, Alonso M, Engelman DM, DiMaio D: Mapping the homodimer interface of an optimized, artificial, transmembrane protein activator of the human erythropoietin receptor. PLoS One. 2014; 2014 Apr 30. PMID: 24788775
- Andreev OA, Engelman DM, Reshetnyak YK: Targeting diseased tissues by pHLIP insertion at low cell surface pH. Front Physiol. 2014; 2014 Mar 13. PMID: 24659971
- Kohlway A, Pirakitikulr N, Barrera FN, Potapova O, Engelman DM, Pyle AM, Lindenbach BD: Hepatitis C virus RNA replication and virus particle assembly require specific dimerization of the NS4A protein transmembrane domain. J Virol. 2014 Jan; 2013 Oct 30. PMID: 24173222
- Fendos J, Barrera FN, Engelman DM: Aspartate embedding depth affects pHLIP's insertion pKa. Biochemistry. 2013 Jul 9; 2013 Jun 27. PMID: 23721379
- Barrera FN, Fendos J, Engelman DM: Membrane physical properties influence transmembrane helix formation. Proc Natl Acad Sci U S A. 2012 Sep 4; 2012 Aug 20. PMID: 22908237
- Barrera FN, Weerakkody D, Anderson M, Andreev OA, Reshetnyak YK, Engelman DM: Roles of carboxyl groups in the transmembrane insertion of peptides. J Mol Biol. 2011 Oct 21; 2011 Aug 23. PMID: 21888917
- Matthews EE, Thévenin D, Rogers JM, Gotow L, Lira PD, Reiter LA, Brissette WH, Engelman DM: Thrombopoietin receptor activation: transmembrane helix dimerization, rotation, and allosteric modulation. FASEB J. 2011 Jul; 2011 Mar 14. PMID: 21402716
- An M, Wijesinghe D, Andreev OA, Reshetnyak YK, Engelman DM: pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation. Proc Natl Acad Sci U S A. 2010 Nov 23; 2010 Nov 3. PMID: 21048084
- Patel PV, Gianoulis TA, Bjornson RD, Yip KY, Engelman DM, Gerstein MB: Analysis of membrane proteins in metagenomics: networks of correlated environmental features and protein families. Genome Res. 2010 Jul; 2010 Apr 29. PMID: 20430783
- Musial-Siwek M, Karabadzhak A, Andreev OA, Reshetnyak YK, Engelman DM: Tuning the insertion properties of pHLIP. Biochim Biophys Acta. 2010 Jun; 2009 Sep 18. PMID: 19766589
- Cammett TJ, Jun SJ, Cohen EB, Barrera FN, Engelman DM, Dimaio D: Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor. Proc Natl Acad Sci U S A. 2010 Feb 23; 2010 Feb 8. PMID: 20142506
- Arinaminpathy Y, Khurana E, Engelman DM, Gerstein MB: Computational analysis of membrane proteins: the largest class of drug targets. Drug Discov Today. 2009 Dec; 2009 Sep 3. PMID: 19733256
- Talbert-Slagle K, Marlatt S, Barrera FN, Khurana E, Oates J, Gerstein M, Engelman DM, Dixon AM, Dimaio D: Artificial transmembrane oncoproteins smaller than the bovine papillomavirus E5 protein redefine sequence requirements for activation of the platelet-derived growth factor beta receptor. J Virol. 2009 Oct; 2009 Jul 15. PMID: 19605488
- Thévenin D, An M, Engelman DM: pHLIP-mediated translocation of membrane-impermeable molecules into cells. Chem Biol. 2009 Jul 31. PMID: 19635412
- Zoonens M, Reshetnyak YK, Engelman DM: Bilayer interactions of pHLIP, a peptide that can deliver drugs and target tumors. Biophys J. 2008 Jul; 2008 Mar 21. PMID: 18359793
- Dupuy AD, Engelman DM: Protein area occupancy at the center of the red blood cell membrane. Proc Natl Acad Sci U S A. 2008 Feb 26; 2008 Feb 19. PMID: 18287056
- Yip KY, Patel P, Kim PM, Engelman DM, McDermott D, Gerstein M: An integrated system for studying residue coevolution in proteins. Bioinformatics. 2008 Jan 15; 2007 Dec 1. PMID: 18056067
- Reshetnyak YK, Segala M, Andreev OA, Engelman DM: A monomeric membrane peptide that lives in three worlds: in solution, attached to, and inserted across lipid bilayers. Biophys J. 2007 Oct 1; 2007 Jun 8. PMID: 17557792
- Matthews EE, Zoonens M, Engelman DM: Dynamic helix interactions in transmembrane signaling. Cell. 2006 Nov 3. PMID: 17081964
- Dixon AM, Stanley BJ, Matthews EE, Dawson JP, Engelman DM: Invariant chain transmembrane domain trimerization: a step in MHC class II assembly. Biochemistry. 2006 Apr 25. PMID: 16618111
- Reshetnyak YK, Andreev OA, Lehnert U, Engelman DM: Translocation of molecules into cells by pH-dependent insertion of a transmembrane helix. Proc Natl Acad Sci U S A. 2006 Apr 25; 2006 Apr 11. PMID: 16608910
- Sachs JN, Engelman DM: Introduction to the membrane protein reviews: the interplay of structure, dynamics, and environment in membrane protein function. Annu Rev Biochem. 2006. PMID: 16756508
- Engelman DM: Membranes are more mosaic than fluid. Nature. 2005 Dec 1. PMID: 16319876
- Chin CN, Sachs JN, Engelman DM: Transmembrane homodimerization of receptor-like protein tyrosine phosphatases. FEBS Lett. 2005 Jul 4. PMID: 15978577
- Freeman-Cook LL, Edwards AP, Dixon AM, Yates KE, Ely L, Engelman DM, Dimaio D: Specific locations of hydrophilic amino acids in constructed transmembrane ligands of the platelet-derived growth factor beta receptor. J Mol Biol. 2005 Jan 28. PMID: 15588835
- Schneider D, Engelman DM: Motifs of two small residues can assist but are not sufficient to mediate transmembrane helix interactions. J Mol Biol. 2004 Oct 29. PMID: 15476801
- Freeman-Cook LL, Dixon AM, Frank JB, Xia Y, Ely L, Gerstein M, Engelman DM, DiMaio D: Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor beta receptor. J Mol Biol. 2004 May 14. PMID: 15111056
- Lehnert U, Xia Y, Royce TE, Goh CS, Liu Y, Senes A, Yu H, Zhang ZL, Engelman DM, Gerstein M: Computational analysis of membrane proteins: genomic occurrence, structure prediction and helix interactions. Q Rev Biophys. 2004 May. PMID: 15999419
- Mitra K, Ubarretxena-Belandia I, Taguchi T, Warren G, Engelman DM: Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol. Proc Natl Acad Sci U S A. 2004 Mar 23; 2004 Mar 11. PMID: 15016920
- Schneider D, Engelman DM: Involvement of transmembrane domain interactions in signal transduction by alpha/beta integrins. J Biol Chem. 2004 Mar 12; 2003 Dec 16. PMID: 14681217
- Liu Y, Gerstein M, Engelman DM: Transmembrane protein domains rarely use covalent domain recombination as an evolutionary mechanism. Proc Natl Acad Sci U S A. 2004 Mar 9; 2004 Mar 1. PMID: 14993608
- Engelman DM, Chen Y, Chin CN, Curran AR, Dixon AM, Dupuy AD, Lee AS, Lehnert U, Matthews EE, Reshetnyak YK, Senes A, Popot JL: Membrane protein folding: beyond the two stage model. FEBS Lett. 2003 Nov 27. PMID: 14630331
- Fisher LE, Engelman DM, Sturgis JN: Effect of detergents on the association of the glycophorin a transmembrane helix. Biophys J. 2003 Nov. PMID: 14581210
- Curran AR, Engelman DM: Sequence motifs, polar interactions and conformational changes in helical membrane proteins. Curr Opin Struct Biol. 2003 Aug. PMID: 12948770
- Dawson JP, Melnyk RA, Deber CM, Engelman DM: Sequence context strongly modulates association of polar residues in transmembrane helices. J Mol Biol. 2003 Aug 1. PMID: 12875850
- Schneider D, Engelman DM: GALLEX, a measurement of heterologous association of transmembrane helices in a biological membrane. J Biol Chem. 2003 Jan 31; 2002 Nov 21. PMID: 12446730
- Engelman DM: Electrostatic fasteners hold the T cell receptor-CD3 complex together. Mol Cell. 2003 Jan. PMID: 12535514
- Schneider D, Liu Y, Gerstein M, Engelman DM: Thermostability of membrane protein helix-helix interaction elucidated by statistical analysis. FEBS Lett. 2002 Dec 4. PMID: 12459496
- Liu Y, Engelman DM, Gerstein M: Genomic analysis of membrane protein families: abundance and conserved motifs. Genome Biol. 2002 Sep 19; 2002 Sep 19. PMID: 12372142
- Mitra K, Steitz TA, Engelman DM: Rational design of 'water-soluble' bacteriorhodopsin variants. Protein Eng. 2002 Jun. PMID: 12082167
- Dawson JP, Weinger JS, Engelman DM: Motifs of serine and threonine can drive association of transmembrane helices. J Mol Biol. 2002 Feb 22. PMID: 11866532