Decrypting the chaperone code

Abstract

The ongoing COVID pandemic has disrupted the ability to hold scientific conferences in person. Despite this, over 200 people from across the world attended The First International Symposium on the Chaperone Code, which was held virtually on October 28 to 29, 2020. The meeting highlighted the many ways that posttranslational modifications (PTMs) on molecular chaperones regulate their function to control proteostasis in diverse organisms. Cells are continuously exposed to a variety of internal and external stressors that induce protein misfolding. To respond and recover from these stresses, cells express a myriad of molecular chaperone and co-chaperone paralogs that aid in folding, refolding, stabilization, activation, and transport of a large proportion of the proteome (“clients”). While chaperones have been extensively studied for over 40 years, the majority of studies have focused on chaperone actions, ATPase cycle regulation, and in vitro folding mechanisms. With the exponential improvement in proteomic technologies over the past 10 years, a huge number of PTMs have been uncovered on chaperones including phosphorylation, acetylation, methylation, SUMOylation, and ubiquitination. Despite the identification of these sites, the respective roles and roles of the modifications (collectively known as the Chaperone Code, Fig. 1) are poorly understood. The inaugural meeting on the Chaperone Code brought together experts from diverse fields ranging from chaperone mechanisms to signal transduction to discuss their latest exciting insights on the Chaperone Code. In this report, we highlight several of these findings in this emerging field of chaperone biology. Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding, stability, and activity of more than 300 proteins also known as clients. Many of these clients are involved in various maladies including cancer, neurodegenerative and infectious diseases (1Schopf F.H. Biebl M.M. Buchner J. The HSP90 chaperone machinery.Nat. Rev. Mol. Cell Biol. 2017; 18: 345-360Crossref PubMed Scopus (475) Google Scholar, 2Woodford M.R. Dunn D.M. Ciciarelli J.G. Beebe K. Neckers L. Mollapour M. Targeting Hsp90 in non-cancerous maladies.Curr. Top Med. Chem. 2016; 16: 2792-2804Crossref PubMed Scopus (9) Google Scholar). Therefore, targeting Hsp90 in preclinical studies and clinical trials in these diseases has been actively pursued (3Neckers L. Workman P. Hsp90 molecular chaperone inhibitors: Are we there yet?.Clin. Cancer Res. 2012; 18: 64-76Crossref PubMed Scopus (685) Google Scholar). Hsp90 chaperone function is linked to its intrinsic ATPase activity of Hsp90. This activity is enhanced by the co-chaperone Aha1 (1Schopf F.H. Biebl M.M. Buchner J. The HSP90 chaperone machinery.Nat. Rev. Mol. Cell Biol. 2017; 18: 345-360Crossref PubMed Scopus (475) Google Scholar). Len Neckers (National Cancer Institute, USA) showed that phosphorylation of a highly conserved Tyr313 in the middle domain of human Hsp90α is important for initial binding of Aha1 (4Xu W. Beebe K. Chavez J.D. Boysen M. Lu Y. Zuehlke A.D. Keramisanou D. Trepel J.B. Prodromou C. Mayer M.P. Bruce J.E. Gelis I. Neckers L. Hsp90 middle domain phosphorylation initiates a complex conformational program to recruit the ATPase-stimulating cochaperone Aha1.Nat. Commun. 2019; 10: 2574Crossref PubMed Scopus (11) Google Scholar). Phosphorylation of Tyr313 appears to provide a phosphorylation-sensitive conformational switch that initiates Aha1 C-domain recruitment to the Hsp90 middle (M) domain and consequent stimulation of ATPase activity. This binding pose of the Aha1 C-domain to Hsp90 M-domain, which was unexpected based on previous models, is supported by recently reported orthogonal cryo-EM data (4Xu W. Beebe K. Chavez J.D. Boysen M. Lu Y. Zuehlke A.D. Keramisanou D. Trepel J.B. Prodromou C. Mayer M.P. Bruce J.E. Gelis I. Neckers L. Hsp90 middle domain phosphorylation initiates a complex conformational program to recruit the ATPase-stimulating cochaperone Aha1.Nat. Commun. 2019; 10: 2574Crossref PubMed Scopus (11) Google Scholar). Further, since tyrosine phosphorylation of Aha1 also facilitates its binding to human Hsp90 (5Dunn D.M. Woodford M.R. Truman A.W. Jensen S.M. Schulman J. Caza T. Remillard T.C. Loiselle D. Wolfgeher D. Blagg B.S. Franco L. Haystead T.A. Daturpalli S. Mayer M.P. Trepel J.B. et al.c-Abl mediated tyrosine phosphorylation of Aha1 activates its co-chaperone function in cancer cells.Cell Rep. 2015; 12: 1006-1018Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), the cooperative action of these PTMs and their impact on protein interaction warrants further investigation. PTMs of Hsp90 can impact its ATPase activity, suggesting an allosteric regulation mechanism in Hsp90. Giorgio Colombo (University of Pavia, Italy) demonstrated the use of computational design in deciphering the chaperone code. Using this approach, it is possible to identify regulatory amino acids that are subject to PTMs and consequently impact the conformational dynamics and ATPase activity of Hsp90 both in vitro and in vivo (6Rehn A. Moroni E. Zierer B.K. Tippel F. Morra G. John C. Richter K. Colombo G. Buchner J. Allosteric regulation points control the conformational dynamics of the molecular chaperone Hsp90.J. Mol. Biol. 2016; 428: 4559-4571Crossref PubMed Scopus (38) Google Scholar). This information can ultimately be used to generate small molecule modulators of function or protein–protein interaction inhibitors. The Hsp90 paralog mitochondrial chaperone tumor necrosis factor receptor-associated protein-1 (TRAP1) is instrumental in metabolic regulation and cell survival. Andrea Rasola, (University of Padova, Italy) showed context-dependent PTMs of TRAP1. More specifically, mutation and inactivation of neurofibromin, a Ras GTPase-activating protein, induce an oncogenic metabolic switch via mitochondrial ERK-mediated phosphorylation of the molecular chaperone TRAP1 (7Masgras I. Ciscato F. Brunati A.M. Tibaldi E. Indraccolo S. Curtarello M. Chiara F. Cannino G. Papaleo E. Lambrughi M. Guzzo G. Gambalunga A. Pizzi M. Guzzardo V. Rugge M. et al.Absence of neurofibromin induces an oncogenic metabolic switch via mitochondrial ERK-mediated phosphorylation of the chaperone TRAP1.Cell Rep. 2017; 18: 659-672Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Dimitra Bourboulia (SUNY Upstate Medical University, USA) discussed how secreted kinase signaling could impact extracellular Hsp90 (eHsp90) chaperone function. She showed that the first secretory eHsp90 co-chaperone TIMP2 (8Baker-Williams A.J. Hashmi F. Budzynski M.A. Woodford M.R. Gleicher S. Himanen S.V. Makedon A.M. Friedman D. Cortes S. Namek S. Stetler-Stevenson W.G. Bratslavsky G. Bah A. Mollapour M. Sistonen L. et al.Co-chaperones TIMP2 and AHA1 competitively regulate extracellular HSP90: Client MMP2 activity and matrix proteolysis.Cell Rep. 2019; 28: 1894-1906.e1896Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), also an endogenous inhibitor of angiogenesis and regulator of MMP2 activity, is phosphorylated by secreted c-Src tyrosine kinase (9Sanchez-Pozo J. Baker-Williams A.J. Woodford M.R. Bullard R. Wei B. Mollapour M. Stetler-Stevenson W.G. Bratslavsky G. Bourboulia D. Extracellular phosphorylation of TIMP-2 by secreted c-Src tyrosine kinase controls MMP-2 activity.iScience. 2018; 1: 87-96Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). This modification could define TIMP2 function as an eHsp90 co-chaperone or an MMP inhibitor. Previous studies have also identified a role for the chaperone code in fungal virulence (10Weissman Z. Pinsky M. Wolfgeher D.J. Kron S.J. Truman A.W. Kornitzer D. Genetic analysis of Hsp70 phosphorylation sites reveals a role in Candida albicans cell and colony morphogenesis.Biochim. Biophys. Acta. 2020; 1868: 140135Crossref Scopus (8) Google Scholar). Stephanie Diezmann (University of Bristol, UK) described a new phosphorylation site (S530) on Candida albicans Hsp90, targeted by casein kinase 2 (CK2), which results in the inhibition of Hsp90 function and blocking expression of key virulence traits. She showed that the phosphomimetic mutant S530E (but not S530A) abolished C. albicans survival at high temperatures, supported a switch to filamentous growth, a morphological change that is important for fungal virulence, and rendered C. albicans susceptible to both antifungal (fluconazole) and Hsp90 (radicicol) drugs (11Alaalm L. Crunden J.L. Butcher M. Obst U. Whealy R. Williamson C.E. O’Brien H.E. Schaffitzel C. Ramage G. Diezmann S. A novel Hsp90 phospho-switch modulates virulence in the major human fungal pathogen Candida albicans.bioRxiv. 2020; https://doi.org/10.1101/2020.09.23.309831Crossref Scopus (0) Google Scholar). These studies support the idea that specific Hsp90 PTMs may be exploited as potential antifungal drug targets. The molecular chaperone heat shock protein 70 (Hsp70) is involved in folding, stability, and quality control of proteins (12Mayer M.P. Gierasch L.M. Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones.J. Biol. Chem. 2019; 294: 2085-2097Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 13Rosenzweig R. Nillegoda N.B. Mayer M.P. Bukau B. The Hsp70 chaperone network.Nat. Rev. Mol. Cell Biol. 2019; 20: 665-680Crossref PubMed Scopus (179) Google Scholar). Hsp70 is also highly regulated by a range of PTMs (14Nitika Porter C.M. Truman A.W. Truttmann M.C. Post-translational modifications of Hsp70 family proteins: Expanding the chaperone code.J. Biol. Chem. 2020; 295: 10689-10708Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Dalia Barsyte-Lovejoy (University of Toronto, Canada) described how characterization of a novel inhibitor of the arginine methylase PRMT7 led to the discovery that Hsp70 is methylated on R469, a highly conserved amino acid present on the client-binding domain. Intriguingly, this PRMT7-catalyzed methylation can only occur when Hsp70 is in its ATP-bound “open” conformation. R469 methylation appears to be important for a wide variety of chaperone processes that include stress granule response to proteasome inhibition and the general response to heat stress (15Szewczyk M.M. Ishikawa Y. Organ S. Sakai N. Li F. Halabelian L. Ackloo S. Couzens A.L. Eram M. Dilworth D. Fukushi H. Harding R. Dela Sena C.C. Sugo T. Hayashi K. et al.Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response.Nat. Commun. 2020; 11: 2396Crossref PubMed Scopus (14) Google Scholar). Lysine methylation comes in several “flavors,” and Magnus Jakobsson (Lund University, Sweden) discussed the impacts of mono, di, and trimethylation of Hsp70 K561 promoted by METTL21A. The trimethylated K561 Hsp70 may have the greatest functional relevance, as it is the major form found in both the nucleus and the cytosol (16Jakobsson M.E. Moen A. Falnes P.O. Correspondence: On the enzymology and significance of HSPA1 lysine methylation.Nat. Commun. 2016; 7: 11464Crossref PubMed Scopus (9) Google Scholar). Functionally, Hsp70 methylation tunes the interaction of Hsp70 with the disease-associated protein alpha-synuclein (17Jakobsson M.E. Moen A. Bousset L. Egge-Jacobsen W. Kernstock S. Melki R. Falnes P.O. Identification and characterization of a novel human methyltransferase modulating Hsp70 protein function through lysine methylation.J. Biol. Chem. 2013; 288: 27752-27763Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Finally, Jakobsson revealed that lysine methylation on Hsp70 in ovarian cancer tumor effusions may be correlated with disease prognosis (18Jakobsson M.E. Moen A. Davidson B. Falnes P.O. Hsp70 (HSPA1) lysine methylation status as a potential prognostic factor in metastatic high-grade serous carcinoma.PLoS One. 2015; 10e0140168Crossref PubMed Scopus (11) Google Scholar). These fascinating results bolster the growing evidence that multiple mutually exclusive modifications of a single Hsp70 residue can uniquely fine-tune chaperone function and represent biomarkers with clinical utility. Hsp70 is intimately tied to oxidative stress and metabolic processes in the cell. Adeleye Afolayan (Medical College of Wisconsin, USA) demonstrated a novel role of Hsp70 in the mitochondrial import of the superoxide dismutase-2 (SOD2, MnSOD) appropriate to the levels of ROS inside the mitochondria. This process is driven by AKT1-catalyzed phosphorylation of Hsp70 on S631, which alters Hsp70 structure and ability to bind the E3 ubiquitin ligase, CHIP. Several avenues of investigation remain to be explored, including the role of adjacent Hsp70 phosphorylation sites S633 and T636, which are also phosphorylated by AKT1 but do not impact the mitochondrial import of SOD2 (19Zemanovic S. Ivanov M.V. Ivanova L.V. Bhatnagar A. Michalkiewicz T. Teng R.J. Kumar S. Rathore R. Pritchard Jr., K.A. Konduri G.G. Afolayan A.J. Dynamic phosphorylation of the C terminus of Hsp70 regulates the mitochondrial import of SOD2 and redox balance.Cell Rep. 2018; 25: 2605-2616.e2607Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Several talks in the symposium described large-scale structural rearrangements on chaperones in response to PTM addition. Richard Bayliss (University of Leeds, UK) described the innovative use of expanded genetic codes in bacteria to produce recombinant Hsp70 phosphorylated on S66. This phosphorylated form was crystallized and revealed that S66 plays a role in Hsp70 interdomain communication. At the cellular level, this Nek6-mediated phosphorylation of Hsp70 is required to maintain a functional mitotic spindle, although the key clients involved are yet to be determined (20Mukherjee M. Sabir S. O'Regan L. Sampson J. Richards M.W. Huguenin-Dezot N. Ault J.R. Chin J.W. Zhuravleva A. Fry A.M. Bayliss R. Mitotic phosphorylation regulates Hsp72 spindle localization by uncoupling ATP binding from substrate release.Sci. Signal. 2018; 11eaao2464Crossref PubMed Scopus (7) Google Scholar). Lila Gierasch (University of Massachusetts Amherst, USA) tested the hypothesis that functionally relevant sites of modifications on Hsp70 chaperone impact its allosteric mechanism. One important site identified was T495 on mammalian Hsp70, a site previously identified in yeast as a functional hotspot as well as being activated in mammalian cells by the legionella pneumophila kinase LegK4 as part of its program of infection (21Moss S.M. Taylor I.R. Ruggero D. Gestwicki J.E. Shokat K.M. Mukherjee S. A Legionella pneumophila kinase phosphorylates the Hsp70 chaperone family to inhibit eukaryotic protein synthesis.Cell Host Microbe. 2019; 25: 454-462.e456Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). She is currently exploring the native regulation of this site in yeast and mammalian cells in collaboration with Andrew Truman (University of North Carolina at Charlotte, USA). Cells have evolved to express a multitude of Hsp70 chaperone paralogs (13Rosenzweig R. Nillegoda N.B. Mayer M.P. Bukau B. The Hsp70 chaperone network.Nat. Rev. Mol. Cell Biol. 2019; 20: 665-680Crossref PubMed Scopus (179) Google Scholar, 22Lotz S.K. Knighton L.E. Nitika Jones G.W. Truman A.W. Not quite the SSAme: Unique roles for the yeast cytosolic Hsp70s.Curr. Genet. 2019; 65: 1127-1134Crossref PubMed Scopus (12) Google Scholar). Several of these are located at spatially distinct sites. An important example is the ER-resident Hsp70 BiP (also known as Grp78), which is essential for the folding of membrane proteins and those with redox-related modifications such as disulfide bonds. Seema Mattoo (Purdue University, USA) described how Huntingtin yeast interacting protein (HYPE; also called FicD) is able to AMPylate the ER-resident Hsp70 BiP at T366 and T518 impacting the BiP ATP cycle. In contrast to many of the other talks demonstrating activation of chaperones through addition of PTMs, her message was that site-specific AMPylation has differential effects on BiP’s ATPase activity and provides a way for cells to stall BiP function in preparation for future stresses (23Sanyal A. Chen A.J. Nakayasu E.S. Lazar C.S. Zbornik E.A. Worby C.A. Koller A. Mattoo S. A novel link between Fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response.J. Biol. Chem. 2015; 290: 8482-8499Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 24Sanyal A. Zbornik E.A. Watson B.G. Christoffer C. Ma J. Kihara D. Mattoo S. Kinetic and structural parameters governing fic-mediated adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78.bioRxiv. 2018; https://doi.org/10.1101/494930Crossref Scopus (0) Google Scholar). Following on from this, Matthias Truttmann (University of Michigan, USA) provided evidence that although AMPylation of BiP in C. elegans does not alter animal survival, it does reduce Aβ toxicity, an effect that can be mimicked by directly silencing Hsp70 chaperones Hsp1, 3, and 4 simultaneously (25Truttmann M.C. Pincus D. Ploegh H.L. Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides.Proc. U. S. A. 2018; PubMed Scopus (11) Google Scholar). the of chaperone code has focused on the Hsp90 and Hsp70 it is that there are many other important of this code. One such example is the heat shock factor family of which regulate the expression of chaperones and a variety of cellular stresses S.V. Sistonen L. insights cellular Cell 2019; PubMed Scopus (9) Google Scholar). Sistonen University, discussed the PTMs of exploring their ability to both and Using a that at a H. K. of 2016; 11: PubMed Scopus Google Scholar), showed that also to highly and expression heat shock A. T. Sistonen L. response to stress is by and Commun. 2017; PubMed Scopus Google Scholar). chaperone PTM studies have focused on phosphorylation, methylation, and acetylation, there are many more that may be important to cells to respond to cellular One such PTM is directly regulated through the between molecular chaperones and poorly it is that of chaperones and may be The of protein is regulated by the of the and the that and to or amino of USA) on the role of in stress She showed that and modification by and to oxidative levels in an This is a example of how PTM such as can be used by cells to and respond to stress N. Y. G.W. The A. J.D. P. G.W. M. T. of Scholar). Buchner discussed the of chaperone code in the the as the of a chaperone to other Buchner that the for that proteins are to this by showed that in the in a chaperone or proteins are and that the mutant proteins do not in the the mutant in protein and the of this that the in the proteome results in as the for than the aggregation of the This ultimately in to or its C. B. F. Richter B. J. C. M. S. T. J. Buchner J. in the proteome are linked to Biol. PubMed Scopus Google Scholar). Andrew Truman (University of North Carolina at Charlotte, USA) the meeting by the of the roles of PTMs in A.W. K. Wolfgeher D. N. S. H. S. Prodromou C. Jones G.W. Kron S.J. Hsp70 phosphorylation controls and 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). by the present of on the Chaperone Code, that major advances in the identification of the myriad of PTMs on than have been multiple levels of future Chaperone Code the first of which is the identification of the stresses and that regulate specific sites on as well as the functional vitro and in of these The is more the between sites and functional In the of the Code, are do sites of PTM on chaperones over and chaperone paralogs have evolved to have At the level, there was of how of this information can be the of the Chaperone Code (14Nitika Porter C.M. Truman A.W. Truttmann M.C. Post-translational modifications of Hsp70 family proteins: Expanding the chaperone code.J. Biol. Chem. 2020; 295: 10689-10708Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). the of the the called a program of collaboration from Chaperone Code and the of Chaperone Code to a and of to the field The that have of with the of this The of the First International Symposium on The Chaperone Truman and Mollapour are to of Upstate Medical for this are also to Lila Gierasch (University of Massachusetts Amherst, USA) for and of this This was supported by the of Medical of the of and W. and The is the of the and does not represent the of the of

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