Molecular Chaperone Research Articles

Proteomic profile of stallion seminal plasma

Proteins mediate all physiological changes. Seminal plasma (SP), the product of testes, epididymis, and accessory sex glands, is a fluid released during ejaculation and represents up to 98% of the voluminous stallion ejaculate. The SP plays an essential role in reproduction as a transit medium and a source of energy, antioxidants, enzymes, and minerals. This study aimed to investigate proteomic profile of the seminal plasma of stallions. Twenty-four Criollo stallions with a known reproductive history and per-cycle concept rate data calculated from at least 30 inseminations were used. One ejaculate was collected from each stallion during the breeding season. Pregnancy rates ranged at day 16 after artificial insemination from 20.2% to 95.6%. The animals were divided into two groups: High Pregnancy (HP), with a pregnancy rate per cycle ≥51%, and Low Pregnancy (LP), with a pregnancy rate of <50%. Sperm concentration, kinetics, morphology, and plasma membrane integrity and functionality were evaluated. The sample was centrifuged at 400xg for 10min. SP was then transferred to a 2 mL microcentrifuge tube and centrifuged again (10.000xg, 60min, 4°C). Proteins were separated using 2D SDS PAGE gel electrophoresis in duplicate. Spots in at least 80% of the gels in one of the groups, with significant abundance (P<0.05) and at least a 1.5-fold magnitude difference between groups, were selected. Thespectra were acquired in a nano LC-MS/MS spectrometer, and proteins were identified by the MASCOT application and validated by SCAFFOLD. From a total of 716 analyzed spots, four were selected. For non-parametric data the Kruskal Wallis and for parametric data Anova and t-test were used. The cysteine-rich secretory protein 3 (CRISP3) and SP protein HSP-1-like (HSP1) showed higher abundance in the SP in group LP than HP (P< 0.05). However, kallikrein-1E2 (KLK) and phospholipase A2 (PLA2) showed higher abundance in HP than LP (P<0.05). In the horse, CRISP-3 is synthesized in great amounts in the accessory sexual glands, ampulla and seminal vesicle. HSP1 has an essential role as a molecular chaperone for sperm motility and viability, in the formation of the oviductal sperm reservoir, in the regulation of cell volume, and possibly in the interaction between gametes. KLK is one of the major proteins found in seminal equine plasma. The kallikreins are serine proteases, and the glandular kallikrein is regulated by androgens and secreted by the prostate. The PLA2 plays an essential role in the late maturational events of spermatozoa, the acrosomal reaction, and sperm egg fusion. In conclusion, the proteins are essential to the biological processes related to stallion fertility, providing insight into SP proteins that could serve as biomarkers for fertility in stallions.

Overexpression of Aha1 and FKBP52 does not affect learning or mood‐like behavior in human tau (hTau) mice

AbstractBackgroundTau plays a critical role in neurodegeneration, thus targeting this protein to control its accumulation is important to prevent or delay neurotoxicity in tauopathies like Alzheimer’s disease (AD). Discrete molecular chaperones, like Aha1 and FKBP52, are known to affect the stability of tau by promoting its phosphorylation, aggregation and, in some mouse models, neuronal death. However, one limitation of preclinical tau models is that various contain tau mutations which do not represent most AD cases. Our study is important because it investigates biological mechanisms and behavioral phenotype in a humanized tau (hTau) mouse model absent of tau mutations. We hypothesize that overexpression of Aha1 in in hTau mice will accelerate tau tangle formation and cognitive deficits.MethodTo test this, Tau KO and hTau mice were bilaterally injected into the hippocampus with AAV9‐Aha1, AAV9‐FKBP52, and AAV9‐mKate (control) virus. At 11 and 13 months of age, animals were tested for cognitive and behavioral phenotypes.ResultsWe found that overexpression of these chaperones does not affect anxiety, spatial working memory, and locomotion in hTau mice. We further confirmed that short‐term working memory in the learning and extinction of fear memories remained unaltered by the high levels of Aha1 and FKBP52 at these ages. Ongoing analyses will examine tau pathology in these brains by measuring tau levels, neuronal density, and cell death markers.ConclusionAt this timepoint, Aha1 and FKBP52 did not induce behavioral differences in these mice. We recently reported that overexpression of these chaperones promotes tau pathological changes in 16‐months‐old wild‐type. Thus, it is possible that behavioral differences can be detected as tau pathology progresses at an older age.

Chaperone Recycling in Late-Stage Flagellar Assembly

The flagellum is a sophisticated nanomachine responsible for motility in Gram-negative bacteria. Flagellar assembly is a strictly choreographed process, in which the motor and export gate are formed first, followed by the extracellular propeller structure. Extracellular flagellar components are escorted to the export gate by dedicated molecular chaperones for secretion and self-assembly at the apex of the emerging structure. The detailed mechanisms of chaperone-substrate trafficking at the export gate remain poorly understood. Here, we structurally characterized the interaction of Salmonella enterica late-stage flagellar chaperones FliT and FlgN with the export controller protein FliJ. Previous studies showed that FliJ is absolutely required for flagellar assembly since its interaction with chaperone-client complexes controls substrate delivery to the export gate. Our biophysical and cell-based data show that FliT and FlgN bind FliJ cooperatively, with high affinity and on specific sites. Chaperone binding completely disrupts the FliJ coiled-coil structure and alters its interactions with the export gate. We propose that FliJ aids the release of substrates from the chaperone and forms the basis of chaperone recycling during late-stage flagellar assembly.

Targeting Sigma-1 Receptor: A Promising Strategy in the Treatment of Parkinson's Disease.

Parkinson's disease is a neurodegenerative disease affecting mainly the elderly population. It is characterized by the loss of dopaminergic neurons of the substantia nigra pars compacta region. Parkinson's disease patients exhibit motor symptoms like tremors, rigidity, bradykinesia/hypokinesia, and non-motor symptoms like depression, cognitive decline, delusion, and pain. Major pathophysiological factors which contribute to neuron loss include excess/misfolded alpha-synuclein aggregates, microglial cell-mediated neuroinflammation, excitotoxicity, oxidative stress, and defective mitochondrial function. Sigma-1 receptors are molecular chaperones located at mitochondria-associated ER membrane. Their activation (by endogenous ligands or agonists) has shown neuroprotective and neurorestorative effects in various diseases. This review discusses the roles of activated Sig-1 receptors in modulating various pathophysiological features of Parkinson's disease like alpha-synuclein aggregates, neuroinflammation, excitotoxicity, and oxidative stress.

LSDDB: Lysosomal Storage Disorder Database for Lysosomal Proteins and Their Single Amino-Acid Substitutions

Lysosomal storage diseases (LSDs) consist of about 60 different monogenic disorders. Most of them occur due to protein misfolding. Only a few of those have been treated with molecular chaperones; the remaining either have limited treatment options or only management therapies. About 1860 single amino-acid substitutions (SAS) have been identified under LSDs. Merely, a handful of mutations have been studied experimentally. Availability of computational tools has made researchers turn toward genetic disorders to focus light on unexplored disorders and their mutations. Since all the LSDs are rare genetic disorders, not much research is carried out in this area. However, a mutational effect on protein function could be predicted, through bioinformatics tools. On that note, out of 1860 SAS, 58 predictions were neutral and 778 were predicted to be disease associated by all programs included in this study. The result of the prediction analysis of all mutations in each of the LSDs is curated into a database. This would make researchers know the deleterious nature of a mutation causing LSD. The database is available at http://lsddb.vit.ac.in:3000/ .

Comparative evaluation of protective immunity against Francisella tularensis induced by subunit or adenovirus-vectored vaccines.

Tularemia is a highly contagious disease caused by infection with Francisella tularensis (Ft), a pathogenic intracellular gram-negative bacterium that infects a wide range of animals and causes severe disease and death in people, making it a public health concern. Vaccines are the most effective way to prevent tularemia. However, there are no Food and Drug Administration (FDA)-approved Ft vaccines thus far due to safety concerns. Herein, three membrane proteins of Ft, Tul4, OmpA, and FopA, and a molecular chaperone, DnaK, were identified as potential protective antigens using a multifactor protective antigen platform. Moreover, the recombinant DnaK, FopA, and Tul4 protein vaccines elicited a high level of IgG antibodies but did not protect against challenge. In contrast, protective immunity was elicited by a replication-defective human type 5 adenovirus (Ad5) encoding the Tul4, OmpA, FopA, and DnaK proteins (Ad5-Tul4, Ad5-OmpA, Ad5-FopA, and Ad5-DnaK) after a single immunization, and all Ad5-based vaccines stimulated a Th1-biased immune response. Moreover, intramuscular and intranasal vaccination with Ad5-Tul4 using the prime-boost strategy effectively eliminated Ft lung, spleen and liver colonization and provided nearly 80% protection against intranasal challenge with the Ft live vaccine strain (LVS). Only intramuscular, not intranasal vaccination, with Ad5-Tul4 protected mice from intraperitoneal challenge. This study provides a comprehensive comparison of protective immunity against Ft provided by subunit or adenovirus-vectored vaccines and suggests that mucosal vaccination with Ad5-Tul4 may yield desirable protective efficacy against mucosal infection, while intramuscular vaccination offers greater overall protection against intraperitoneal tularemia.

Hsp90 mutants with distinct defects provide novel insights into cochaperone regulation of the folding cycle.

Molecular chaperones play a key role in maintaining proteostasis and cellular health. The abundant, essential, cytosolic Hsp90 (Heat shock protein, 90 kDa) facilitates the folding and activation of hundreds of newly synthesized or misfolded client proteins in an ATP-dependent folding pathway. In a simplified model, Hsp70 first helps load client onto Hsp90, ATP binding results in conformational changes in Hsp90 that result in the closed complex, and then less defined events result in nucleotide hydrolysis, client release and return to the open state. Cochaperones bind and assist Hsp90 during this process. We previously identified a series of yeast Hsp90 mutants that appear to disrupt either the 'loading', 'closing' or 'reopening' events, and showed that the mutants had differing effects on activity of some clients. Here we used those mutants to dissect Hsp90 and cochaperone interactions. Overexpression or deletion of HCH1 had dramatically opposing effects on the growth of cells expressing different mutants, with a phenotypic shift coinciding with formation of the closed conformation. Hch1 appears to destabilize Hsp90-nucleotide interaction, hindering formation of the closed conformation, whereas Cpr6 counters the effects of Hch1 by stabilizing the closed conformation. Hch1 and the homologous Aha1 share some functions, but the role of Hch1 in inhibiting progression through the early stages of the folding cycle is unique. Sensitivity to the Hsp90 inhibitor NVP-AUY922 also correlates with the conformational cycle, with mutants defective in the loading phase being most sensitive and those defective in the reopening phase being most resistant to the drug. Overall, our results indicate that the timing of transition into and out of the closed conformation is tightly regulated by cochaperones. Further analysis will help elucidate additional steps required for progression through the Hsp90 folding cycle and may lead to new strategies for modulating Hsp90 function.

Elucidation of the Binding Mechanism of Anionic Phospholipids to Antioxidant Protein Peroxiredoxin 2.

Peroxiredoxins (Prxs) belong to a family of ubiquitously expressed peroxidases that detoxify reactive oxygen species. In addition to their enzymatic function, Prxs also function as molecular chaperones. This functional switch is related to their degree of oligomerization. We have previously revealed that Prx2 interacts with anionic phospholipids and that the anionic phospholipid-containing Prx2 oligomer forms a high molecular weight (HMW) complex in a nucleotide-dependent manner. However, the detailed mechanism of the oligomer and HMW complex formation remains unclear. In this study, we investigated the anionic phospholipid binding site in Prx2 using site-directed mutagenesis to understand the mechanism of the oligomer formation. Our findings demonstrated that six binding site residues in Prx2 are important for the binding of anionic phospholipids.

Germline C1GALT1C1 mutation causes a multisystem chaperonopathy

Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase (C1GALT1), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal C1GALT1C1. Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in C1GALT1C1. They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type C1GALT1C1 partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients.

Insulin-like growth factor binding protein-2 and glucose-regulated protein 78 kDa: Potential biomarkers affect prognosis in IDH-wildtype glioblastoma patients.

The overall survival of IDH-wildtype glioblastoma patients is poor despite best available treatments. There is an urgent need for new biomarkers to inform more precise disease stratification. Previous studies have identified insulin-like growth factor binding protein-2 (IGFBP-2) as a potential biomarker for glioblastoma diagnosis and therapeutic targeting. Other studies have indicated links between the insulin-like growth factor (IGF) axis and tumorigenic functions of a molecular chaperone glucose related protein of 78 kDa (GRP78). We aimed to interrogate the oncogenic effects of IGFBP-2 and GRP78 in our glioma stem cell (GSC) lines and clinical cohort. Immunoblotting, reverse transcription quantitative real-time PCR were used to quantify protein and mRNA levels derived from GSCs and non-malignant neural stem cells (NSCs). Microarray analysis was used to compare the differences in IGFBP-2 (IGFBP-2) and GRP78 (HSPA5) transcript expression between NSCs, GSCs and adult human cortex samples. Immunohistochemistry was used to quantify IGFBP-2 and GRP78 expression in IDH-wildtype glioblastoma tissue sections (n = 92) and clinical implications assessed using survival analysis. Finally, the relationship between IGFBP-2 and GRP78 was further explored molecularly using coimmunoprecipitation. Here, we demonstrate that IGFBP-2 and HSPA5 mRNA is overexpressed in GSCs and NSCs in comparison to non-malignant brain tissue. We also determined a relationship in which G144 and G26 GSCs expressed higher IGFBP-2 protein and mRNA than GRP78, and this was reversed in mRNA isolated from adult human cortex samples. Clinical cohort analysis revealed that Glioblastomas with high IGFBP-2 protein expression paired with low GRP78 protein expression were significantly associated with a much shorter survival time (Median = 4 months, p = 0.019) compared with 12-14 months for any other combination of high/low protein expression. Inverse levels of IGFBP-2 and GRP78 may be adverse clinical prognostic markers in IDH-wildtype glioblastoma. Further interrogation of the mechanistic link between IGFBP-2 and GRP78 may be important for rationalisation of their potential as biomarkers and therapeutic targets.

Functional characterization of the DNA-binding protein from starved cells (DPS) as a molecular chaperone under heat stress

The DNA-binding protein from starved cells, known as DPS, has been characterized as a crucial factor in protecting E. coli from external stresses. The DPS functions in various cellular processes, including protein-DNA binding, ferroxidase activity, compaction of chromosome and regulation of stress resistance gene expression. DPS proteins exist as oligomeric complexes; however, the specific biochemical activity of oligomeric DPS in conferring heat shock tolerance has not been fully understood. Therefore, we investigated the novel functional role of DPS under heat shock. To elucidate the functional role of DPS under heat shock conditions, we purified recombinant GST-DPS protein and demonstrated its thermostability and existence in its highly oligomeric form. Furthermore, we discovered that the hydrophobic region of GST-DPS influenced the formation of oligomers, which exhibited molecular chaperone activity, thereby preventing the aggregation of substrate proteins. Collectively, our findings highlight the novel functional role of DPS, as a molecular chaperone and may confer thermotolerance to E. coli.

Improved Recombinant Expression of Maltogenic α-Amylase AmyM in Bacillus subtilis by Optimizing Its Secretion and NADPH Production

The maltose α-amylase AmyM from Bacillus stearothermophilus can be used for flour modification, baked goods preservation, and maltose production. Here, we optimized the recombinant expression of AmyM in Bacillus subtilis WB800 via several strategies. By screening the optimal promoter, a double promoter combination (P43 and PamyL) could improve the expression level of AmyM by 61.25%, compared with the strong promoter P43. Then, we optimized the secretion efficiency of recombinant AmyM by over-expressing the molecular chaperone prsA gene. SDS-PAGE results suggested that over-expression of the prsA could improve the secretion efficiency of AmyM to the extracellular environment. The extracellular enzyme activity of AmyM was increased by 101.58% compared to the control strain. To further improve the expression of AmyM, we introduced the hemoglobin gene of Vitreoscilla (vgb) into the AmyM recombinant strain. The results revealed that the introduction of vgb could promote the transcription and translation of AmyM in B. subtilis. This may be due to the increasing level of intracellular NADPH and NADP+ caused by the expression of vgb. By this strategy, the expression level of AmyM was increased by 204.08%. Finally, we found the recombinant AmyM showed an optimal temperature of 65 °C and an optimal pH of 5.5. Our present results provided an effective strategy for increasing the heterologous expression level of AmyM in B. subtilis.

In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies.

The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface-binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection. IMPORTANCE With the currently available antibiotics, the treatment of TB takes months. The slow response to treatment is caused by antibiotic tolerance, which is especially common among bacteria that form biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of antibiotics. To be able to develop faster-acting treatments against TB, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterize the protein composition of Mmr biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first sybodies that bind to mycobacterial biofilms. As nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target therapies to biofilms.

A new kid in the folding funnel: Molecular chaperone activities of the BRICHOS domain.

The BRICHOS protein superfamily is a diverse group of proteins associated with a wide variety of human diseases, including respiratory distress, COVID-19, dementia, and cancer. A key characteristic of these proteins-besides their BRICHOS domain present in the ER lumen/extracellular part-is that they harbor an aggregation-prone region, which the BRICHOS domain is proposed to chaperone during biosynthesis. All so far studied BRICHOS domains modulate the aggregation pathway of various amyloid-forming substrates, but not all of them can keep denaturing proteins in a folding-competent state, in a similar manner as small heat shock proteins. Current evidence suggests that the ability to interfere with the aggregation pathways of substrates with entirely different end-point structures is dictated by BRICHOS quaternary structure as well as specific surface motifs. This review aims to provide an overview of the BRICHOS protein family and a perspective of the diverse molecular chaperone-like functions of various BRICHOS domains in relation to their structure and conformational plasticity. Furthermore, we speculate about the physiological implication of the diverse molecular chaperone functions and discuss the possibility to use the BRICHOS domain as a blood-brain barrier permeable molecular chaperone treatment of protein aggregation disorders.

Molecular chaperones in stroke-induced immunosuppression.

Stroke-induced immunosuppression is a process that leads to peripheral suppression of the immune system after a stroke and belongs to the central nervous system injury-induced immunosuppressive syndrome. Stroke-induced immunosuppression leads to increased susceptibility to post-stroke infections, such as urinary tract infections and stroke-associated pneumonia, worsening prognosis. Molecular chaperones are a large class of proteins that are able to maintain proteostasis by directing the folding of nascent polypeptide chains, refolding misfolded proteins, and targeting misfolded proteins for degradation. Various molecular chaperones have been shown to play roles in stroke-induced immunosuppression by modulating the activity of other molecular chaperones, cochaperones, and their associated pathways. This review summarizes the role of molecular chaperones in stroke-induced immunosuppression and discusses new approaches to restore host immune defense after stroke.

A first-in-class inhibitor of Hsp110 molecular chaperones of pathogenic fungi

Proteins of the Hsp110 family are molecular chaperones that play important roles in protein homeostasis in eukaryotes. The pathogenic fungus Candida albicans, which causes infections in humans, has a single Hsp110, termed Msi3. Here, we provide proof-of-principle evidence supporting fungal Hsp110s as targets for the development of new antifungal drugs. We identify a pyrazolo[3,4-b] pyridine derivative, termed HLQ2H (or 2H), that inhibits the biochemical and chaperone activities of Msi3, as well as the growth and viability of C. albicans. Moreover, the fungicidal activity of 2H correlates with its inhibition of in vivo protein folding. We propose 2H and related compounds as promising leads for development of new antifungals and as pharmacological tools for the study of the molecular mechanisms and functions of Hsp110s.

HSP90α is needed for the survival of rod photoreceptors and regulates the expression of rod PDE6 subunits

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the stability of a small set of proteins essential in various cellular pathways. Cytosolic HSP90 has two closely related paralogs: HSP90α and HSP90β. Due to the structural and sequence similarities of cytosolic HSP90 paralogs, identifying the unique functions and substrates in the cell remains challenging. In this article, we assessed the role of HSP90α in the retina using a novel HSP90α murine knockout model. Our findings show that HSP90α is essential for rod photoreceptor function but was dispensable in cone photoreceptors. In the absence of HSP90α, photoreceptors developed normally. We observed rod dysfunction in HSP90α knockout at 2months with the accumulation of vacuolar structures, apoptotic nuclei, and abnormalities in the outer segments. The decline in rod function was accompanied by progressive degeneration of rod photoreceptors that was complete at 6months. The deterioration in cone function and health was a "bystander effect" that followed the degeneration of rods. Tandem mass tag proteomics showed that HSP90α regulates the expression levels of <1% of the retinal proteome. More importantly, HSP90α was vital in maintaining rod PDE6 and AIPL1 cochaperone levels in rod photoreceptor cells. Interestingly, cone PDE6 levels were unaffected. The robust expression of HSP90β paralog in cones likely compensates for the loss of HSP90α. Overall, our study demonstrated the critical need for HSP90α chaperone in the maintenance of rod photoreceptors and showed potential substrates regulated by HSP90α in the retina.

Activation of XBP1 but not ATF6α rescues heart failure induced by persistent ER stress in medaka fish.

The unfolded protein response is triggered in vertebrates by ubiquitously expressed IRE1α/β (although IRE1β is gut-specific in mice), PERK, and ATF6α/β, transmembrane-type sensor proteins in the ER, to cope with ER stress, the accumulation of unfolded and misfolded proteins in the ER. Here, we burdened medaka fish, a vertebrate model organism, with ER stress persistently from fertilization by knocking out the AXER gene encoding an ATP/ADP exchanger in the ER membrane, leading to decreased ATP concentration-mediated impairment of the activity of Hsp70- and Hsp90-type molecular chaperones in the ER lumen. ER stress and apoptosis were evoked from 4 and 6 dpf, respectively, leading to the death of all AXER-KO medaka by 12 dpf because of heart failure (medaka hatch at 7 dpf). Importantly, constitutive activation of IRE1α signaling-but not ATF6α signaling-rescued this heart failure and allowed AXER-KO medaka to survive 3 d longer, likely because of XBP1-mediated transcriptional induction of ER-associated degradation components. Thus, activation of a specific pathway of the unfolded protein response can cure defects in a particular organ.

Transcriptional regulation of Sis1 promotes fitness but not feedback in the heat shock response.

The heat shock response (HSR) controls expression of molecular chaperones to maintain protein homeostasis. Previously, we proposed a feedback loop model of the HSR in which heat-denatured proteins sequester the chaperone Hsp70 to activate the HSR, and subsequent induction of Hsp70 deactivates the HSR (Krakowiak et al., 2018; Zheng et al., 2016). However, recent work has implicated newly synthesized proteins (NSPs) - rather than unfolded mature proteins - and the Hsp70 co-chaperone Sis1 in HSR regulation, yet their contributions to HSR dynamics have not been determined. Here, we generate a new mathematical model that incorporates NSPs and Sis1 into the HSR activation mechanism, and we perform genetic decoupling and pulse-labeling experiments to demonstrate that Sis1 induction is dispensable for HSR deactivation. Rather than providing negative feedback to the HSR, transcriptional regulation of Sis1 by Hsf1 promotes fitness by coordinating stress granules and carbon metabolism. These results support an overall model in which NSPs signal the HSR by sequestering Sis1 and Hsp70, while induction of Hsp70 - but not Sis1 - attenuates the response.

The ribosome quality control factor Asc1 determines the fate of HSP70 mRNA on and off the ribosome.

Cells survive harsh environmental conditions by potently upregulating molecular chaperones such as heat shock proteins (HSPs), particularly the inducible members of the HSP70 family. The life cycle of HSP70 mRNA in the cytoplasm is unique-it is translated during stress when most cellular mRNA translation is repressed and rapidly degraded upon recovery. Contrary to its 5' untranslated region's role in maximizing translation, we discovered that the HSP70 coding sequence (CDS) suppresses its translation via the ribosome quality control (RQC) mechanism. The CDS of the most inducible Saccharomyces cerevisiae HSP70 gene, SSA4, is uniquely enriched with low-frequency codons that promote ribosome stalling during heat stress. Stalled ribosomes are recognized by the RQC components Asc1p and Hel2p and two novel RQC components, the ribosomal proteins Rps28Ap and Rps19Bp. Surprisingly, RQC does not signal SSA4 mRNA degradation via No-Go-Decay. Instead, Asc1p destabilizes SSA4 mRNA during recovery from heat stress by a mechanism independent of ribosome binding and SSA4 codon optimality. Therefore, Asc1p operates in two pathways that converge to regulate the SSA4 mRNA life cycle during stress and recovery. Our research identifies Asc1p as a critical regulator of the stress response and RQC as the mechanism tuning HSP70 synthesis.