A small heat shock protein, GmHSP17.9, from nodule confers symbiotic nitrogen fixation and seed yield in soybean.

Legume-rhizobia symbiosis enables biological nitrogen fixation to improve crop production for sustainable agriculture. Small heat shock proteins (sHSPs) are involved in multiple environmental stresses and plant development processes. However, the role of sHSPs in nodule development in soybean remains largely unknown. In the present study, we identified a nodule-localized sHSP, called GmHSP17.9, in soybean, which was markedly up-regulated during nodule development. GmHSP17.9 was specifically expressed in the infected regions of the nodules. GmHSP17.9 overexpression and RNAi in transgenic composite plants and loss of function in CRISPR-Cas9 gene-editing mutant plants in soybean resulted in remarkable alterations in nodule number, nodule fresh weight, nitrogenase activity, contents of poly ß-hydroxybutyrate bodies (PHBs), ureide and total nitrogen content, which caused significant changes in plant growth and seed yield. GmHSP17.9 was also found to act as a chaperone for its interacting partner, GmNOD100, a sucrose synthase in soybean nodules which was also preferentially expressed in the infected zone of nodules, similar to GmHSP17.9. Functional analysis of GmNOD100 in composite transgenic plants revealed that GmNOD100 played an essential role in soybean nodulation. The hsp17.9 lines showed markedly more reduced sucrose synthase activity, lower contents of UDP-glucose and acetyl coenzyme A (acetyl-CoA), and decreased activity of succinic dehydrogenase (SDH) in the tricarboxylic acid (TCA) cycle in nodules due to the missing interaction with GmNOD100. Our findings reveal an important role and an unprecedented molecular mechanism of sHSPs in nodule development and nitrogen fixation in soybean.

Small Heat Shock Proteins Collaborate with FAIM to Prevent Accumulation of Misfolded Protein Aggregates.

Cells and tissues are continuously subject to environmental insults, such as heat shock and oxidative stress, which cause the accumulation of cytotoxic, aggregated proteins. We previously found that Fas Apoptosis Inhibitory Molecule (FAIM) protects cells from stress-induced cell death by preventing abnormal generation of protein aggregates similar to the effect of small heat shock proteins (HSPs). Protein aggregates are often associated with neurodegenerative diseases, including Alzheimer's disease (AD). In this study, we sought to determine how FAIM protein dynamics change during cellular stress and how FAIM prevents the formation of amyloid-ß aggregates/fibrils, one of the pathological hallmarks of AD. Here, we found that the majority of FAIM protein shifts to the detergent-insoluble fraction in response to cellular stress. A similar shift to the insoluble fraction was also observed in small heat shock protein (sHSP) family molecules, such as HSP27, after stress. We further demonstrate that FAIM is recruited to sHSP-containing complexes after cellular stress induction. These data suggest that FAIM might prevent protein aggregation in concert with sHSPs. In fact, we observed the additional effect of FAIM and HSP27 on the prevention of protein aggregates using an in vitro amyloid-ß aggregation model system. Our work provides new insights into the interrelationships among FAIM, sHSPs, and amyloid-ß aggregation.

Lysine acetylation of Hsp16.3: Effect on its structure, chaperone function and influence towards the growth of Mycobacterium tuberculosis.

Hsp16.3 plays a vital role in the slow growth of Mycobacterium tuberculosis via its chaperone function. Many secretory proteins, including Hsp16.3 undergo acetylation in vivo. Seven lysine (K) residues (K64, K78, K85, K114, K119, K132 and K136) in Hsp16.3 are acetylated inside pathogen. However, how lysine acetylation affects its structure, chaperone function and pathogen's growth is still elusive. We examined these aspects by executing in vitro chemical acetylation (acetic anhydride modification) and by utilizing a lysine acetylation mimic mutant (Hsp16.3-K64Q/K78Q/K85Q/K114Q/K119Q/K132Q/K136Q). Far- and near-UV CD measurements revealed that the chemically acetylated proteins(s) and acetylation mimic mutant has altered secondary and tertiary structure than unacetylated/wild-type protein. The chemical modification and acetylation mimic mutation also disrupted the oligomeric assembly, increased surface hydrophobicity and reduced stability of Hsp16.3, as revealed by GF-HPLC, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid binding and urea denaturation experiments, respectively. These structural changes collectively led to an enhancement in chaperone function (aggregation and thermal inactivation prevention ability) of Hsp16.3. Moreover, when the H37Rv strain expressed the acetylation mimic mutant protein, its growth was slower in comparison to the strain expressing the wild-type/unacetylated Hsp16.3. Altogether, these findings indicated that lysine acetylation improves the chaperone function of Hsp16.3 which may influence pathogen's growth in host environment.

Small Heat Shock Protein (sHsp22.98) from Trialeurodes vaporariorum Plays Important Role in Apple Scar Skin Viroid Transmission.

Trialeurodes vaporariorum, commonly known as the greenhouse whitefly, severely infests important crops and serves as a vector for apple scar skin viroid (ASSVd). This vector-mediated transmission may cause the spread of infection to other herbaceous crops. For effective management of ASSVd, it is important to explore the whitefly's proteins, which interact with ASSVd RNA and are thereby involved in its transmission. In this study, it was found that a small heat shock protein (sHsp) from T. vaporariorum, which is expressed under stress, binds to ASSVd RNA. The sHsp gene is 606 bp in length and encodes for 202 amino acids, with a molecular weight of 22.98 kDa and an isoelectric point of 8.95. Intermolecular interaction was confirmed through in silico analysis, using electrophoretic mobility shift assays (EMSAs) and northwestern assays. The sHsp22.98 protein was found to exist in both monomeric and dimeric forms, and both forms showed strong binding to ASSVd RNA. To investigate the role of sHsp22.98 during ASSVd infection, transient silencing of sHsp22.98 was conducted, using a tobacco rattle virus (TRV)-based virus-induced gene silencing system. The sHsp22.98-silenced whiteflies showed an approximate 50% decrease in ASSVd transmission. These results suggest that sHsp22.98 from T. vaporariorum is associated with viroid RNA and plays a significant role in transmission.

A Nodule-Localized Small Heat Shock Protein GmHSP17.1 Confers Nodule Development and Nitrogen Fixation in Soybean.

Small heat shock proteins (sHSPs) are ubiquitous proteins present in all organisms. The sHSPs are not only upregulated under heat shock as well as other stresses but also are expressed in unstressed cells, indicating quite diverse functions of sHSPs. However, there is little known about the role of sHSPs in nodulation and nitrogen fixation in soybean. In this study, we cloned a candidate protein of sHSP, GmHSP17.1, from proteome of nodule and analyzed its function in soybean nodulation. We found that GmHSP17.1 was a cytosolic protein and preferentially expressed during nodule development. An overexpression of GmHSP17.1 in composite transgenic plants showed increases in nodule number, fresh weight, nodule size, area of infection cells, and nitrogenase activity, and subsequently promoted the content of nitrogen and growth of soybean plants. While GmHSP17.1 RNA interference (RNAi) lines showed significantly impaired nodule development and nitrogen fixation efficiency. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS), GmRIP1 was identified as the first potential target of GmHSP17.1, and was shown to be specifically expressed in soybean nodules. The interaction between GmHSP17.1 and GmRIP1 was further confirmed by yeast-two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) in vivo and pull-down assay in vitro. Furthermore, peroxidase activity was markedly increased in GmHSP17.1 overexpressed nodules and decreased in RNAi lines. As a result, the reactive oxygen species (ROS) content greatly decreased in GmHSP17.1 overexpression lines and increased in suppression lines. Taken together, we conclude that GmHSP17.1 plays an important role in soybean nodulation through interacting with GmRIP1. Our results provide foundation for studying the mechanism of nitrogen fixation and for the genetics improvement of legume plants.

Nucleosomal association and altered interactome underlie the mechanism of cataract caused by the R54C mutation of αA-crystallin.

BACKGROUND: αA-crystallin plays an important role in eye lens development. Its N-terminal domain is implicated in several important biological functions. Mutations in certain conserved arginine residues in the N-terminal region of αA-crystallin lead to cataract with characteristic cytoplasmic/nuclear aggregation of the mutant protein. In this study, we attempt to gain mechanistic insights into the congenital cataract caused by the R54C mutation in human αA-crystallin. METHODS: We used several spectroscopic techniques to investigate the structure and function of the wild-type and R54CαA-crystallin. Immunoprecipitation, chromatin-enrichment followed by western blotting, immunofluorescence and cell-viability assay were performed to study the interaction partners, chromatin-association, stress-like response and cell-death caused by the mutant. RESULTS: Although R54CαA-crystallin exhibited slight changes in quaternary structure, its chaperone-like activity was comparable to that of wild-type. When expressed in lens epithelial cells, R54CαA-crystallin exhibited a speckled appearance in the nucleus rather than cytoplasmic localization. R54CαA-crystallin triggered a stress-like response, resulting in nuclear translocation of αB-crystallin, disassembly of cytoskeletal elements and activation of caspase 3, leading to apoptosis. Analysis of the "interactome" revealed an increase in interaction of the mutant protein with nucleosomal histones, and its association with chromatin. CONCLUSIONS: The study shows that alteration of "interactome" and nucleosomal association, rather than loss of chaperone-like activity, is the molecular basis of cataract caused by the R54C mutation in αA-crystallin. GENERAL SIGNIFICANCE: The study provides a novel mechanism of cataract caused by a mutant of αA-crystallin, and sheds light on the possible mechanism of stress and cell death caused by such nuclear inclusions.

The Small Ones Matter-sHsps in the Bacterial Chaperone Network.

Small heat shock proteins (sHsps) are an evolutionarily conserved class of ATP-independent chaperones that form the first line of defence during proteotoxic stress. sHsps are defined not only by their relatively low molecular weight, but also by the presence of a conserved α-crystallin domain, which is flanked by less conserved, mostly unstructured, N- and C-terminal domains. sHsps form oligomers of different sizes which deoligomerize upon stress conditions into smaller active forms. Activated sHsps bind to aggregation-prone protein substrates to form assemblies that keep substrates from irreversible aggregation. Formation of these assemblies facilitates subsequent Hsp70 and Hsp100 chaperone-dependent disaggregation and substrate refolding into native species. This mini review discusses what is known about the role and place of bacterial sHsps in the chaperone network.

Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize.

ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5), deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs), late embryogenesis abundant (LEA) proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation.

The potential role of small heat shock proteins in mitochondria.

Mitochondria play a central role in cellular metabolism, calcium homeostasis, redox signaling and cell fates. Mitochondrial homeostasis is tightly regulated, and mitochondrial dysfunction is frequently associated with severe human pathologies. Small heat shock proteins are molecular chaperones that play major roles in development, stress responses, and diseases, and have been envisioned as targets for therapy. The mechanisms that lie behind the cytoprotection of small heat shock proteins are related to the regulation of mitochondrial functions. This review recapitulates the current knowledge of the expression of various small heat shock proteins in mitochondria and discusses their implication in the role of mitochondria and their regulation. Based on their involvement in mitochondrial normal physiology and pathology, a better understanding of their roles and regulation will pave the way for innovative approaches for the successful treatment of a range of stress-related syndromes whose etiology is based upon dysfunction of mitochondria.