Biology subjects

Mayer, M. P.

Publications and source records attributed to Mayer, M. P..

9 recordsLinked to original sources

bioRxiv2024

Modification of regulatory tyrosines biases human Hsp90α for interaction with cochaperones and clients

The highly conserved Hsp90 chaperones control stability and activity of many essential signaling and regulatory proteins including many protein kinases, E3 ligases and transcription factors. Thereby, Hsp90s couple cellular homeostasis of the proteome to cell fate decisions. High-throughput mass spectrometry revealed 178 and 169 posttranslational modifications (PTMs) for human cytosolic Hsp90 and Hsp90{beta}, but for only a few of the modifications the physiological consequences are investigated in some detail. In this study, we explored the suitability of the yeast model system for the identification of key regulatory residues in human Hsp90. Replacement of three tyrosine residues known to be phosphorylated by phosphomimetic glutamate and by non-phosphorylatable phenylalanine individually and in combination influenced yeast growth and the maturation of 7 different Hsp90 clients in distinct ways. Furthermore, wild-type and mutant Hsp90 differed in their ability to stabilize known clients when expressed in HepG2 HSP90AA1-/- cells. The purified mutant proteins differed in their interaction with the cochaperones Aha1, Cdc37, Hop and p23 and in their support of the maturation of glucocorticoid receptor ligand binding domain in vitro. In vivo and in vitro data correspond well to each other confirming that the yeast system is suitable for the identification of key regulatory sites in human Hsp90s. Our findings indicate that even closely related clients are affected differently by the amino acid replacements in the investigated positions, suggesting that PTMs could bias Hsp90s client specificity.

molecular biology↗

bioRxiv2021

Induction of mitochondrial heat shock proteins and mitochondrial biogenesis in endothelial cells upon acute methylglyoxal stress: Evidence for hormetic autofeedback

Increased metabolic flux produces potentially harmful side-products, such as reactive dicarbonyl and oxygen species. The reactive dicarbonly methylglyoxal (MG) can impair oxidative capacity, which is downregulated in type 2 diabetes. Heat shock proteins (HSPs) of subfamily A (Hsp70s) promote ATP-dependent processing of damaged proteins during MG exposure which also involve mitochondrial proteins. Since the protection of mitochondrial proteins could promote higher production of reactive metabolites due to increased substrate flux, tight regulation of HspA-mediated protein handling is important. We hypothesized that stress-inducible HspAs (HspA1A/HspA1B) are pivotal for maintaining mitochondrial biogenesis during acute MG-stress. To analyze the role of stress-inducible HspA1A/HspA1B for maintenance of mitochondrial homeostasis during acute MG exposure, we knocked out HSPA1A/HSPA1B in mouse endothelial cells. HSPA1A/HSPA1B KO cells showed upregulation of the mitochondrial chaperones HspA9 (mitochondrial Hsp70/mortalin) and HspD1 (Hsp60) as well as induction of mitochondrial biogenesis upon MG exposure. Increased mitochondrial biogenesis was reflected by elevated mitochondrial branching, total count and area as well as by upregulation of mitochondrial proteins and corresponding transcription factors. Our findings suggest that mitochondrial HspA9 and HspD1 promote mitochondrial biogenesis during acute MG stress, which is counterregulated by HspA1A/HspA1B to prevent mitochondrial overstimulation and to maintain balanced oxidative capacity under metabolic stress conditions. These data support an important role of HSPs in MG-induced hormesis.

cell biology↗

bioRxiv2021

Direct observation of Hsp90-induced compaction in a protein chain

The chaperone Hsp90 is well known to undergo important conformational changes, which depend on nucleotide, co-chaperones, substrate interactions and post-translational modifications. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally, yet central to its function. Here, using optical tweezers and luciferase and glucocorticoid receptor substrates, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable kinases and receptor clients, regulates their activity, and how Hsp90 cooperates with Hsp70.

biophysics↗

bioRxiv2021

Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence

The pathology associated with malaria infection is largely due to the ability of infected human erythrocytes to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. Taken together, our data reveal a role for PFA66 in host cell modification, implicate human HSP70 as also being essential in this process, and uncover a KAHRP-independent mechanism for correct knob biogenesis. Our observations open up exciting new avenues for the development of new anti-malarials.

cell biology↗

bioRxiv2020

Hsf1 is SUMOylated in the activated trimeric state

The heat shock response (HSR) is a transcriptional program of organisms to counteract an imbalance in protein homeostasis. It is orchestrated in all eukaryotic cells by heat shock factor 1 (Hsf1). Despite very intensive research, the intricacies of the Hsf1 activation-attenuation cycle remain elusive at a molecular level. Posttranslational modifications belong to one of the key mechanisms proposed to adapt the Hsf1 activity to the needs of individual cells and phosphorylation of Hsf1 at multiple sites has attracted much attention. According to cell biological and proteomics data, Hsf1 is also modified by SUMO (small ubiquitin-like modifier) at several sites. How SUMOylation affects Hsf1 activity at a molecular level is still unclear. Here, we analyzed Hsf1 SUMOylation in vitro with purified components to address questions that could not be answered in cell culture models. In vitro Hsf1 is primarily conjugated at lysine 298 with a single SUMO, though we did detect low level SUMOylation at other sites. None of the tested E3 SUMO ligases increased SUMOylation efficacy as compared to the level in the presence of high concentrations of the E2 Ubc9. We provide evidence that Hsf1 trimerization and phosphorylation at serines 303 and 307 increases SUMOylation efficiency, suggesting that Hsf1 is SUMOylated in its activated state. Hsf1 can be SUMOylated when DNA-bound, and SUMOylation of Hsf1 does neither alter DNA binding affinity nor does it affect Hsc70 and DnaJB1-mediated monomerization of Hsf1 trimers and concomitant dislocation from DNA. We propose that SUMOylation acts at the transcription level of the HSR.

biochemistry↗

bioRxiv2020

Targeted Proteomics Reveals Quantitative Differences in Low Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation

Protein glycosylation is essential in all domains of life and its mutational impairment in humans can result in severe diseases named Congenital Disorders of Glycosylation (CDGs). Studies on molecular level are however challenging, because many glycosyltransferases in the endoplasmic reticulum (ER) are low abundance membrane proteins. We established a comprehensive multiple reaction monitoring (MRM) assay to quantify most human glycosyltransferases involved in the processes of N-glycosylation,O- and C-mannosylation in the ER. To increase reproducibility, a membrane protein fraction of isotopically labeled HEK 293T cells was used as an internal standard. With this internal standard the MRM assay is easily transferable between laboratories. 22 glycosyltransferases could be reliably quantified from whole cell lysates of HEK 293T cells, HeLa cells and skin fibroblast cell lines. We then analyzed fibroblasts derived from CDG type I patients with mutations in the ALG1,ALG2 or ALG11 gene. Mutations in ALG1 or ALG2 gene strongly reduced the levels of the ALG1 and ALG2 protein, respectively. In contrast, the levels of all other glycosyltransferases remained unchanged, which was unexpected given evidence that the ALG1, ALG2 and ALG11 proteins form a stable complex. This study describes an efficient workflow for the development of MRM assays for low abundance proteins, establishes a ready-to-use tool for the comprehensive quantification of ER-localized glycosyltransferases and provides new insight into the organization of disease-relevant glycosylation processes.

biochemistry↗

bioRxiv2019

Molecular mechanism of attenuation of heat shock transcription factor 1 activity

The heat shock response is a universal transcriptional response to proteotoxic stress orchestrated by heat shock transcription factor Hsf1 in all eukaryotic cells. Despite over 40 years of intense research, the mechanism of HSF1 activity regulation remains poorly understood at a molecular level. In metazoa Hsf1 trimerizes upon heat shock through a leucin-zipper domain and binds to DNA. How Hsf1 is dislodged from DNA and monomerized remained enigmatic. Here, we demonstrate that trimeric Hsf1 is dissociated from DNA in vitro by Hsc70 and DnaJB1. Hsc70 acts at two distinct sites on Hsf1. Hsf1 trimers are monomerized by successive cycles of entropic pulling, unzipping the triple leucine-zipper. This process directly monitors the concentration of Hsc70 and DnaJB1. During heat shock adaptation Hsc70 first binds to the transactivation domain leading to partial attenuation of the response and subsequently, at higher concentrations, Hsc70 removes Hsf1 from DNA to restore the resting state.

molecular biology↗

bioRxiv2018

Refolding activity of bacterial Hsp90 in vivo reveals ancient chaperoning function

The conserved molecular chaperones Hsp70 and Hsp90 play a key role in protein folding. Mechanistically, Hsp90 acts downstream from Hsp70 solving an Hsp70-inflicted folding block. It is unclear, though, when and to which extend the concerted action of this cascade becomes crucial in living organisms. Here we show that, in E. coli cells, Hsp90 dramatically improves protein refolding after heat stress while it is dispensable for de novo folding. We found that Hsp90 inhibition effectively reduced the refolding yields in vivo, leading to strongly reduced enzymatic activity of the paradigmatic chaperone client luciferase and broadly increased aggregation of the E. coli proteome. Additionally, the presence of Hsp90 during refolding reduces the net ATP consumption presumably by sparing the substrate binding-and-release cycles on Hsp70. This mechanism explains how the cooperation of Hsp90 with the Hsp70 chaperone system creates robust folding machinery in a sustainable manner. Together, we describe a general function for bacterial Hsp90 as a key factor of the folding cascade, which may be the ancient activity of this evolutionary conserved machine.

biochemistry↗

bioRxiv2017

Breaking the Deadlock of Molecular Chaperones

Protein folding in the cell requires ATP-driven chaperone machines. It is poorly understood, however, how these machines fold proteins. Here we propose that the conserved Hsp70 and Hsp90 chaperones support formation of the folding nucleus by providing a gradient of decreasing hydrophobicity. Early on the folding pathway Hsp70 uses its highly hydrophobic binding pocket to recover a stalled, unproductive folding intermediate. The aggressive nature of Hsp70 action, however, blocks productive folding by grabbing hydrophobic, core-forming segments. This precludes on-pathway nucleation at high, physiological Hsp70 levels. Transfer to the less hydrophobic Hsp90 enables the intermediate to resume forming its folding nucleus. Subsequently, the protein enters a spontaneous folding trajectory towards its native state, independent of the ATPase activities of both Hsp70 and Hsp90. Our findings provide a general mechanistic concept for chaperoned protein folding.

biochemistry↗