Small Heat Shock Protein Research Articles

Proteomic analysis of dietary restriction in yeast reveals a role for Hsp26 in replicative lifespan extension.

Dietary restriction (DR) has been shown to increase lifespan in organisms ranging from yeast to mammals. This suggests that the underlying mechanisms may be evolutionarily conserved. Indeed, upstream signalling pathways, such as TOR, are strongly linked to DR-induced longevity in various organisms. However, the downstream effector proteins that ultimately mediate lifespan extension are less clear. To shed light on this, we used a proteomic approach on budding yeast. Our reasoning was that analysis of proteome-wide changes in response to DR might enable the identification of proteins that mediate its physiological effects, including replicative lifespan extension. Of over 2500 proteins we identified by liquid chromatography–mass spectrometry, 183 were significantly altered in expression by at least 3-fold in response to DR. Most of these proteins were mitochondrial and/or had clear links to respiration and metabolism. Indeed, direct analysis of oxygen consumption confirmed that mitochondrial respiration was increased several-fold in response to DR. In addition, several key proteins involved in mating, including Ste2 and Ste6, were down-regulated by DR. Consistent with this, shmoo formation in response to α-factor pheromone was reduced by DR, thus confirming the inhibitory effect of DR on yeast mating. Finally, we found that Hsp26, a member of the conserved small heat shock protein (sHSP) family, was up-regulated by DR and that overexpression of Hsp26 extended yeast replicative lifespan. As overexpression of sHSPs in Caenorhabditis elegans and Drosophila has previously been shown to extend lifespan, our data on yeast Hsp26 suggest that sHSPs may be universally conserved effectors of longevity.

New Insights on the Impact of Cattle Handling on Post-Mortem Myofibrillar Muscle Proteome and Meat Tenderization.

This study investigated the effect of different cattle management strategies at farm (Intensive vs. Extensive) and during transport and lairage (mixing vs. non-mixing with unfamiliar animals) on the myofibrillar subproteome of Longissimus thoracis et lumborum (LTL) muscle of “Asturiana de los Valles” yearling bulls. It further aimed to study the relationships with beef quality traits including pH, color, and tenderness evaluated by Warner–Bratzler shear force (WBSF). Thus, comparative proteomics of the myofibrillar fraction along meat maturation (from 2 h to 14 days post-mortem) and different quality traits were analyzed. A total of 23 protein fragments corresponding to 21 unique proteins showed significant differences among the treatments (p < 0.05) due to any of the factors considered (Farm, Transport and Lairage, and post-mortem time ageing). The proteins belong to several biological pathways including three structural proteins (MYBPC2, TNNT3, and MYL1) and one metabolic enzyme (ALDOA) that were affected by both Farm and Transport/Lairage factors. ACTA1, LDB3, and FHL2 were affected by Farm factors, while TNNI2 and MYLPF (structural proteins), PKM (metabolic enzyme), and HSPB1 (small Heat shock protein) were affected by Transport/Lairage factors. Several correlations were found between the changing proteins (PKM, ALDOA, TNNI2, TNNT3, ACTA1, MYL1, and CRYAB) and color and tenderness beef quality traits, indicating their importance in the determination of meat quality and their possible use as putative biomarkers.

Genome wide identification and characterization of small heat shock protein gene family in pigeonpea and their expression profiling during abiotic stress conditions

Small heat shock proteins as large multigene family are present ubiquitously among Archaea to Eukaryota. The sHSPs are molecular chaperones that maintain the proper protein folding and disaggregation of denatured proteins during stress conditions. In the present study, out of identified 38 sHSPs in the pigeonpea genome, the 20 are distributed across seven chromosomes while the remaining are located on unassembled scaffolds. These Cc_sHSPs are classified into 16 subfamilies. The cytoplasmic class-II is the largest sub-family with five Cc_sHSPs. The gene structure analysis revealed that Cc_sHSP genes specifically containing no or very few introns. The promoter analysis revealed the presence of various cis-acting elements responsible for developmental, biotic, and abiotic stress specific-induction of Cc_sHSPs. A total of one segmental duplication and four tandem duplication events are identified for Cc_sHSPs. The qRT-PCR based expression analysis of all 38 Cc_sHSP genes was conducted for diverse abiotic stress conditions. The Cc_sHSP genes are highly induced by heat, drought, cold, and salt stresses indicating a key role in mitigating the various abiotic stress responses. The divergence time of paralogous Cc_sHSPs ranged from 8.66 to 191.82 MYA. The present study can be a strong basis for the functional characterization of Cc_sHSPs.

Delay in puberty is dependent on heat shock protein B1 expression in native cross layers of Punjab under heat stress.

Small heat shock protein B1 (HspB1) has been reported to play an essential role in thermotolerance. This study aimed to determine a correlation (if any) between HspB1 expression and age at first egg lay (puberty) in a native cross-layer poultry of Punjab under heat stress. Forty native cross-layer birds were reared in two different seasons, viz. summer (THI was more than 27), classified as the heat-stressed group (n=20) and winter season (THI was less than 21), classified as the control group (n=20). Blood was collected from both the groups of birds in their 15th week of age and at puberty. Serum catalase and superoxide dismutase activities, reduced glutathione and corticosterone concentration and lipid peroxidation were measured to assess the oxidative stress in both the groups of birds. The serum antioxidants significantly decreased whilst corticosterone levels and lipid peroxidation significantly elevated in birds in response to summer heat stress. Moreover, in summer season, the activities of the antioxidant enzymes further decreased and lipid peroxidation further increased significantly in birds from their pre-pubertal stage to puberty, which was not observed during the winter season. A clone of chicken HspB1 in BL21 (DE3) cells was revived, and recombinant HspB1 was purified using Ni-NTA agarose column. Serum HspB1 concentration was estimated in different groups of birds by indirect ELISA that has been standardized using the recombinant chicken HspB1. Compared to the control, birds under heat stress had significantly higher serum HspB1 levels. The delay in puberty of all the heat-stressed birds was significantly associated with the increase in their serum HspB1 levels. Taken together, the expression of HspB1 was found to be associated with age at puberty in the native cross poultry layers of Punjab.

Transgenic double-stranded RNA rice, a potential strategy for controlling striped stem borer (Chilo suppressalis).

Although the striped stem borer (SSB, Chilo suppressalis Walker) is a devastating pest of rice that causes significant economic losses, management options are currently limited. Plant-mediated RNA interference (RNAi) is an emerging crop protection technique in which transgenic plants are modified to express insect-specific double-stranded RNAs (dsRNAs) that trigger RNAi silencing in target pests. In this study, an RNAi-based screen of 35 candidate SSB genes identified a small heat shock protein gene (CssHsp) as a potential plant-based RNAi target. To assess its utility in planta, a total of 39 transgenic rice plants were generated, with 11 independent transformants found to contain a single copy of the dsCssHsp expression cassette. In life-time feeding bioassays, three transgenic lines (DS10, DS35, DS36) were found to have significant negative impacts on SSB populations. After feeding for 8 days, mortality in the three transgenic lines exceeded 60%. By pupation, mortality further increased to 90% and few SSB survived to eclosion. Gene expression analyses confirmed that CssHsp transcript levels were significantly reduced after feeding on the transgenic dsCssHsp rice. These results demonstrate the potential for developing a plant-mediated RNAi strategy targeting CssHsp as a more biorational field-based approach for SSB control. © 2021 Society of Chemical Industry.

Investigating the secretion of small heat shock proteins in reactive astrocytes in Alzheimer’s disease

AbstractBackgroundMolecular chaperones have protective functions in neurodegeneration by preventing misfolding of aggregation‐prone proteins. These functions have been mostly studied in neurons. However, members of the family of chaperones known as small heat shock proteins (sHSPs), including HSPB1, CRYAB, and HSPB8, are specifically expressed in glial cells rather than in neurons, and their levels increase in astrocytes in human post‐mortem Alzheimer’s disease (AD) brain.While traditionally considered intracellular proteins, novel extracellular functions have been attributed to chaperones in cancer or the immune system. Interestingly, HSPB1 is also found in senile plaques suggesting these can also be extracellular in the brain. We hypothesize that sHSP can be extracellular and aim to investigate whether sHSPs are secreted from astrocytes in Alzheimer’s disease brain.MethodWe used human post‐mortem brain sections from Alzheimer’s disease patients to investigate changes in extracellular/intracellular localization of HSPB1. To further explore sHSPs secretion, we used primary mouse astrocytes. Reactive astrocytes were induced by TNFα and IL‐1α, mimicking the local environment in the diseased brain. We investigated changes in astrocyte cell lysates and the astrocyte conditioned media by Western blot. To investigate the secretion mechanisms, we also use size exclusion chromatography (SEC) and nanoparticle tracking analysis (NTA)ResultAlthough sHSPs are intracellular chaperones, Our analysis of AD patients post‐mortem brain sections suggested that extracellular HSPB1 might increase around astrocytes that are close to amyloid plaques. In primary mouse astrocytes, we also found an increase in sHSPs in the extracellular media when astrocytes become reactive upon treatment with TNFα and IL‐1α. We further investigated their mechanism of secretion by fractionating the astrocyte conditioned media using SEC. NTA and proteinase K protection assays show that the sHSPs were not present in extracellular vesicle fractions, but rather free in the media.ConclusionOur study suggests that HSPB1 is secreted from astrocytes near to plaques in AD brain, as well as from reactive astrocytes in culture, and this secretion is not dependent on extracellular vesicles. The mechanisms behind how sHSPs are secreted and their extracellular functions still need to be explored.

Investigating the non‐cell autonomous role of glial chaperones in Alzheimer’s disease

AbstractBackgroundAstrocytes are vital in the onset and progression of Alzheimer’s disease (AD). Accumulation of reactive astrocytes, together with tau phosphorylation, correlates very strongly with cognitive decline.Molecular chaperones are essential for maintaining protein homeostasis. One family of chaperones are the small heat shock proteins (sHSPs), which include HSPB1 and CRYAB. Expression of these sHSPs seems to be restricted to glial cells, and levels are found to increase in those astrocytes found in AD brains. The role that sHSP play in astrocytes in AD is still not known. Emerging evidence suggests vital interplay between different cell types during neurodegenerative diseases. We therefore aim to investigate the non‐cell autonomous role of astrocytic sHSPs in AD.MethodWe are using primary mouse neurons and organotypic brain slice cultures in conjunction with the recombinant adeno‐associated viral system (rAAV). These systems will allow us to replicate the tau pathology found in AD, investigate neuron‐astrocyte interactions and determine whether either can be altered by overexpression of our sHSPs.Using immunofluorescence, confocal microscopy and western blotting we are characterising expression and localization of sHSPs in post‐mortem human AD brain tissue and brain slice cultures.ResultOur data shows that HSPB1 is specifically expressed in astrocytes in both human brain tissue and slice cultures. Interestingly, HSPB1 levels in the human brain increase in GFAP‐positive astrocytes surrounding amyloid plaques. When organotypic brain slices are treated with cytokines or Aβ oligomers, reactive astrocytes are induced and levels of HSPB1 and CRYAB are also increased, similar to what we observe in human AD brain.ConclusionOur results in human AD brain highlight the importance of astrocytic HSPB1 in AD, and we provide evidence to suggest not only that organotypic brain slices are a good model to replicate and study the function of sHSPs in AD, but that HSPB1 and CRYAB play an important role in the response to AD‐relevant pathology.Using rAAVs, we aim to manipulate astrocytic sHSPs levels in organotypic brain slices to investigate whether astrocytic sHSPs could modulate neuronal health and tau pathology in a non‐cell autonomous manner.

Extracellular vesicle-associated small heat shock proteins as therapeutic agents in neurodegenerative diseases and beyond

Increasing evidence points towards using extracellular vesicles (EVs) as a therapeutic strategy in neurodegenerative diseases such as multiple sclerosis, Parkinson's, and Alzheimer's disease. EVs are nanosized carriers that play an essential role in intercellular communication and cellular homeostasis by transporting an active molecular cargo, including a large variety of proteins. Recent publications demonstrate that small heat shock proteins (HSPBs) exhibit a beneficial role in neurodegenerative diseases. Moreover, it is defined that HSPBs target the autophagy and the apoptosis pathway, playing a prominent role in chaperone activity and cell survival. This review elaborates on the therapeutic potential of EVs and HSPBs, in particular HSPB1 and HSPB8, in neurodegenerative diseases. We conclude that EVs and HSPBs positively influence neuroinflammation, central nervous system (CNS) repair, and protein aggregation in CNS disorders. Moreover, we propose the use of HSPB-loaded EVs as advanced nanocarriers for the future development of neurodegenerative disease therapies.

Small heat shock protein αB-crystallin potentiates Aβ neurotoxicity by hetero-oligomeric stabilization.

Amyloid plaque, a neuropathological hallmark of Alzheimer's disease (AD), is composed of neurotoxic amyloid-β (Aβ) peptides and other proteins, including the small heat shock protein αB-crystallin (also known as HSPB5). We investigated the cellular origin of αB-crystallin, its secretion properties and how it may affect formation of neurotoxic Aβ hetero-oligomers. Immunohistofluorescence, Confocal Microscopy, Immunogold electron microscopy, SDS-page and immunoblotting, electron paramagnetic resonance spectroscopy (EPR), quasi-elastic light scattering (QLS) spectroscopy, cell and organotypic slice culture, LDH and propidium iodide assays. Immunohistofluorescence analysis of human AD brain revealed that Aβ and αB-crystallin co-localize in extracellular dense core and diffuse plaques and in surrounding reactive astrocytes. Immunohistochemical and immunoblotting analyses showed that astrocytes secrete αB-crystallin in vitro via an exosomal pathway. Co-incubation of αB-crystallin and Aβ suppressed Aβ fibrillogenesis by sequestration in hetero-oligomeric complexes however, this resulted in αB-crystallin potentiated Aβ neurotoxicity in mouse primary neurons and organotypic rat hippocampus slice culture as revealed by LDH and Propidium Iodide assays. Moreover, αB-crystallin acutely potentiated Aβ-induced impairment of activity-dependent long-term potentiation (LTP) of rat Schaffer collateral-CA1 synaptic transmission. These results indicate that the small heat shock protein αB-crystallin is secreted by reactive astrocytes and potentiates Aβ neurotoxicity by stabilizing hetero-oligomers.

Structural and functional studies of D109A human αB-crystallin contributing to the development of cataract and cardiomyopathy diseases.

αB-crystallin (heat shock protein β5/HSPB5) is a member of the family of small heat shock proteins that is expressed in various organs of the human body including eye lenses and muscles. Therefore, mutations in the gene of this protein (CRYAB) might have many pathological consequences. A new mutation has recently been discovered in the α-crystallin domain of this chaperone protein which replaces aspartate 109 with alanine (D109A). This mutation can cause myofibrillar myopathy (MFM), cataracts, and cardiomyopathy. In the current study, several spectroscopic and microscopic analyses, as well as gel electrophoresis assessment were applied to elucidate the pathogenic contribution of human αB-crystallin bearing D109A mutation in development of eye lens cataract and myopathies. The protein oligomerization, chaperone-like activity and chemical/thermal stabilities of the mutant and wild-type protein were also investigated in the comparative assessments. Our results suggested that the D109A mutation has a significant impact on the important features of human αB-crystallin, including its structure, size of the protein oligomers, tendency to form amyloid fibrils, stability, and chaperone-like activity. Given the importance of aspartate 109 in maintaining the proper structure of the α-crystallin domain, its role in the dimerization and chaperone-like activity, as well as preserving protein stability through the formation of salt bridges; mutation at this important site might have critical consequences and can explain the genesis of myopathy and cataract disorders. Also, the formation of large light-scattering aggregates and disruption of the chaperone-like activity by D109A mutation might be considered as important contributing factors in development of the eye lens opacity.

Interaction between hypoviral-regulated fungal virulence factor laccase3 and small heat shock protein Hsp24 from the chestnut blight fungus Cryphonectria parasitica.

Laccase3 is an important virulence factor of the fungus Cryphonectria parasitica. Laccase3 gene (lac3) transcription is induced by tannic acid, a group of phenolic compounds found in chestnut trees, and its induction is regulated by the hypovirus CHV1 infection. CpHsp24, a small heat shock protein gene of C. parasitica, plays a determinative role in stress adaptation and pathogen virulence. Having uncovered in our previous study that transcriptional regulation of the CpHsp24 gene in response to tannic acid supplementation and CHV1 infection was similar to that of the lac3, and that conserved phenotypic changes of reduced virulence were observed in mutants of both genes, we inferred that both genes were implicated in a common pathway. Building on this finding, in this paper we examined whether the CpHsp24 protein (CpHSP24) was a molecular chaperone for the lac3 protein (LAC3). Our pull-down experiment indicated that the protein products of the two genes directly interacted with each other. Heterologous co-expression of CpHsp24 and lac3 genes using Saccharomyces cerevisiae resulted in more laccase activity in the cotransformant than in a parental lac3-expresssing yeast strain. These findings suggest that CpHSP24 is, in fact, a molecular chaperone for the LAC3, which is critical component of fungal pathogenesis.

Mitochondria-Targeted SmsHSP24.1 Overexpression Stimulates Early Seedling Vigor and Stress Tolerance by Multi-Pathway Transcriptome-Reprogramming.

Among the diverse array of heat shock proteins across the three domains of life, mitochondria-targeted small heat shock proteins (sHSPs) are evolved in the plant lineage. However, they remained mysterious and understudied. In this study, we reported a systematic study of a novel mitochondria-targeted nuclear sHSP from eggplant (Solanum melongena L.; SmsHSP24.1). Differential expression of SmsHSP24.1 indicated its positive role exerted during stress conditions. Escherichia coli-BL21 cell line overexpressing the SmsHSP24.1 showed excellent thermo-tolerance ability, tolerating up to 52°C. Spectrometry and electron microscopy revealed a multimeric structure of the protein which acted as a molecular chaperone at high temperatures. Overexpression of SmsHSP24.1 significantly enhanced resistance against heat, drought, and salt stresses and showed rapid germination in constitutively overexpressed eggplant lines. RNA-seq analysis reveals an apparent upregulation of a set of reactive oxygen species (ROS) scavenging enzymes of the glutathione (GHS) pathway and mitochondrial electron transport chain (ETC). Significant upregulation was also observed in auxin biosynthesis and cell-wall remodeling transcripts in overexpressed lines. qPCR, biochemical and physiological analysis further aligned with the finding of transcriptome analysis and suggested an essential role of SmsHSP24.1 under various stress responses and positive physiological influence on the growth of eggplants. Therefore, this gene has immense potential in engineering stress-resilient crop plants.

Phosphorylation activates the yeast small heat shock protein Hsp26 by weakening domain contacts in the oligomer ensemble

Hsp26 is a small heat shock protein (sHsp) from S. cerevisiae. Its chaperone activity is activated by oligomer dissociation at heat shock temperatures. Hsp26 contains 9 phosphorylation sites in different structural elements. Our analysis of phospho-mimetic mutations shows that phosphorylation activates Hsp26 at permissive temperatures. The cryo-EM structure of the Hsp26 40mer revealed contacts between the conserved core domain of Hsp26 and the so-called thermosensor domain in the N-terminal part of the protein, which are targeted by phosphorylation. Furthermore, several phosphorylation sites in the C-terminal extension, which link subunits within the oligomer, are sensitive to the introduction of negative charges. In all cases, the intrinsic inhibition of chaperone activity is relieved and the N-terminal domain becomes accessible for substrate protein binding. The weakening of domain interactions within and between subunits by phosphorylation to activate the chaperone activity in response to proteotoxic stresses independent of heat stress could be a general regulation principle of sHsps.

Heat shock in Cronobacter sakazakii induces direct protection and cross-protection against simulated gastric fluid stress

The bacterial heat shock response in foodborne pathogens is caused by exposure to higher temperatures which poses a great threat to food safety because it can undermine food processing interventions and host defense. The study assessed the heat and acid resistance of Cronobacter sakazakii following heat shock (53 °C for 15min). Inactivation curves of the heat-shocked and non-shocked C. sakazakii cells at four temperatures (56, 58, 60, and 62 °C) and simulated gastric fluid (SGF, pH 3.0) were examined and fitted with the log-linear model and the Weibull model. The inactivation parameters obtained on the basis of the Weibull model showed that heat shock significantly (p < 0.05) increased the values of δ (time to reach 1 log reduction) and t3d (time to reach 3 log reduction) under thermal and acid inactivation. The results proved that heat shock provided C. sakazakii direct protection from a more adverse heat challenge and cross-protection from SGF, i.e. there was a heat shock response. Results of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that seven protein species showed enhanced expression, while four protein species showed decreased expression in the heat-shocked compared to the non-heat-shocked C. sakazakii cells. Quantitative real-time reverse transcriptase PCR (RT-qPCR) revealed upregulation of six stress related genes, ibpA, ibpB (both encoding molecular chaperons), Hsp15 (encoding heat shock protein), Hsp20 (encoding small heat-shock protein), HspQ (encoding proteases) and rpoS (encoding stationary phase sigma factor), following heat shock treatment. In addition, heat shock induced an increase proportion of saturated fatty acids (SFA), cyclic fatty acids (CFA) and the ratio of saturated fatty acids to unsaturated fatty acids (SFA/USFA), whereas reducing the proportion of unsaturated fatty acids (USFA). Consequently, establishment of inactivation models of C. sakazakii could provide data support for quantitative microbial risk assessment (QMRA). Exploration of enhanced resistance mechanisms might provide clues for prevention and control of contamination by heat-shocked C. sakazakii.

Ethanol Alleviates Amyloid-β-Induced Toxicity in an Alzheimer's Disease Model of Caenorhabiditis elegans.

Amyloid-β, a hallmark of Alzheimer’s disease, forms toxic intracellular oligomers and extracellular senile plaques resulting in neuronal toxicity. Ethanol is widely consumed worldwide. Moderate ethanol consumption has numerous benefits in humans. We found that ethanol could significantly extend the lifespan of Caenorhabiditis elegans in a previous study. Based on that study, we tested the effect of ethanol on Alzheimer’s disease transgenic Caenorhabiditis elegans strain CL4176, which expresses amyloid-β1-42 peptide in body wall muscle cells. Ethanol delayed paralysis and reduced amyloid-β oligomers in Caenorhabiditis elegans worms of the CL4176 strain. Moreover, ethanol could induce the nuclear translocation of DAF-16 in the nematodes. However, in worms that were fed daf-16 RNAi bacteria, ethanol no longer delayed the paralysis. The qPCR assays showed that ethanol increases the expression of daf-16, hsf-1 and their common target genes- small heat shock protein genes. In addition, we also found that ethanol could increase lysosome mass in the CL4176 worms. In summary, our study indicated that ethanol attenuated amyloid-β toxicity in the Alzheimer’s disease model of Caenorhabiditis elegans via increasing the level of lysosomes to promote amyloid-β degradation and upregulating the levels of small heat shock protein genes to reduce amyloid-β aggregation.

Honey bee sHSP are responsive to diverse proteostatic stresses and potentially promising biomarkers of honey bee stress

The pollination services provided by the honey bee are critical in both natural and agricultural ecosystems. Honey bee colonies in the United States have suffered from an increased rate of die-off in recent years, stemming from a complex set of interacting stresses that remain poorly described. Defining specific common cellular processes and cellular stress responses impacted by multiple stressors represent a key step in understanding these synergies. Proteotoxic stresses negatively impact protein synthesis, folding, and degradation. Diverse proteotoxic stresses induce expression of genes encoding small heat shock proteins (sHSP) of the expanded lethal (2) essential for life (l(2)efl) gene family. In addition to upregulation by the Integrated Stress Response (ISR), the Heat Shock Response (HSR), and the Oxidative Stress Response (OSR), our data provide first evidence that sHSP genes are upregulated by the Unfolded Protein Response (UPR). As these genes appear to be part of a core stress response that could serve as a useful biomarker for cellular stress in honey bees, we designed and tested an RT-LAMP assay to detect increased l(2)efl gene expression in response to heat-stress. While this assay provides a powerful proof of principle, further work will be necessary to link changes in sHSP gene expression to colony-level outcomes, to adapt our preliminary assay into a Point of Care Testing (POCT) assay appropriate for use as a diagnostic tool for use in the field, and to couple assay results to management recommendations.

Loss of Hsp67Bc leads to autolysosome enlargement in the Drosophila brain.

Hsp67Bc is a small heat shock protein found in Drosophila melanogaster. Apart from performing a function (common for all small heat shock proteins) of preventing aggregation of misfolded proteins, it is involved in macroautophagy regulation alongside the Starvin protein. Overexpression of the D. melanogaster Hsp67Bc gene has been shown to stimulate macroautophagy in S2 cell culture. Nonetheless, it has been unknown how the absence of the Hsp67Bc gene may affect it. Here, we studied the effect ofHsp67Bc gene deletion on the macroautophagy induced by the pathogenic Wolbachia wMelPop strain in D. melanogaster. We detected Wolbachia inside autophagic vacuoles in fly neurons, thereby proving that these endosymbionts were being eliminated via macroautophagy. Nevertheless, we did not register any difference in brain bacterial load between Hsp67Bc-null and control flies at all tested stages of ontogenesis. Moreover, the abundance of autophagic vacuoles was similar between neurons of the mutant and control flies, yet the cross-sectional area of autolysosomes on ultrathin sections was more than 1.5-fold larger in Hsp67Bc-null fly brains than in the control line. Our findings suggest that the product of the Hsp67Bc gene does not participate in the initiation of endosymbiont-induced macroautophagy but may mediate autophagosome maturation: the deletion of the Hsp67Bc gene leads to the increase in autolysosome size.

Multiple nanocages of a cyanophage small heat shock protein with icosahedral and octahedral symmetries

The structures of a cyanophage small heat shock protein (sHSP) were determined as octahedrons of 24-mers and 48-mers and as icosahedrons of 60-mers. An N-terminal deletion construct of an 18 kDa sHSP of Synechococcus sp. phage S-ShM2 crystallized as a 24-mer and its structure was determined at a resolution of 7 Å. The negative stain electron microscopy (EM) images showed that the full-length protein is a mixture of a major population of larger and a minor population of smaller cage-like particles. Their structures have been determined by electron cryomicroscopy 3D image reconstruction at a resolution of 8 Å. The larger particles are 60-mers with icosahedral symmetry and the smaller ones are 48-mers with octahedral symmetry. These structures are the first of the viral/phage origin and the 60-mer is the largest and the first icosahedral assembly to be reported for sHSPs.

Archaeal Hsp14 drives substrate shuttling between small heat shock proteins and thermosome: insights into a novel substrate transfer pathway.

Heat shock proteins maintain protein homeostasis and facilitate the survival of an organism under stress. Archaeal heat shock machinery usually consists of only sHsps, Hsp70, and Hsp60. Moreover, Hsp70 is absent in thermophilic and hyperthermophilic archaea. In the absence of Hsp70, how aggregating protein substrates are transferred to Hsp60 for refolding remains elusive. Here, we investigated the crosstalk in the heat shock response pathway of thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. In the present study, we biophysically and biochemically characterized one of the small heat shock proteins, Hsp14, of S. acidocaldarius. Moreover, we investigated its ability to interact with Hsp20 and Hsp60 to facilitate the substrate proteins' folding under stress conditions. Like Hsp20, we demonstrated that the dimer is the active form of Hsp14, and it forms an oligomeric storage form at a higher temperature. More importantly, the dynamics of the Hsp14 oligomer are maintained by rapid subunit exchange between the dimeric states, and the rate of subunit exchange increases with increasing temperature. We also tested the ability of Hsp14 to form hetero-oligomers via subunit exchange with Hsp20. We observed hetero-oligomer formation only at higher temperatures (50 °C-70 °C). Furthermore, experiments were performed to investigate the interaction between small heat shock proteins and Hsp60. We demonstrated an enthalpy-driven direct physical interaction between Hsp14 and Hsp60. Our results revealed that Hsp14 could transfer sHsp-captured substrate proteins to Hsp60, which then refolds them back to their active form.

STAY-GREEN and light-harvesting complex II chlorophyll a/b binding protein are involved in albinism of a novel albino tea germplasm ‘Huabai 1’

‘Huabai 1’ is a new albino tea germplasm, with high amino acids in its albinism period, however, the albino mechanism remains unknown. To understand the albinism mechanism of ‘Huabai 1’, molecular-level studies were carried out to reveal the functions of CsLHCII and CsSGR in albinism. In this study, transmission electron microscopy (TEM) observations revealed that albino leaves of ‘Huabai 1’ are more damaged than that of ‘Baiye 1’. CsSGR overexpression in A. thaliana and N. benthamiana indicated that CsSGR is involved in the regulation of leaf albinism, and it may play an important role as a key regulator for the dismantling of photosynthetic chlorophyll - LHCP complexes. Moreover, CsLHCII overexpression promotes leaf regreening. The Yeast two-hybrid (Y2H) screening identified a chloroplast small heat shock protein (CssHSP, XM_028206028.1) which binds to CsSGR and CsLHCII respectively, forming CsSGR-CssHSP-CsLHCII protein complexes that may function in regulating albinism of ‘Huabai 1’.