High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid.

In conventional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. Genetically incorporating a photoreactive amino acid offers two key advantages: any site can be targeted, including those that are inaccessible to conventional crosslinking reagents, and photoreactive amino acids can potentially react with a broad range of interaction partners. However, broad reactivity imposes additional challenges for crosslink identification. In this study, we incorporate benzoylphenylalanine (BPA), a photoreactive amino acid, at selected sites in an intrinsically disordered region of the human protein HSPB5. We report and characterize a workflow for identifying and visualizing residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, which is up to ten times more than existing tools for residue-level BPA crosslink identification. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between replicate analyses. Based on these results, we anticipate that this workflow will support the more general use of genetically incorporated, photoreactive amino acids for characterizing the structures of proteins that have resisted high-resolution characterization.

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.

Interaction of the C-terminal immunoglobulin-like domains (Ig 22-24) of filamin C with human small heat shock proteins.

Small heat shock proteins are the well-known regulators of the cytoskeleton integrity, yet their complexes with actin-binding proteins are underexplored. Filamin C, a dimeric 560 kDa protein, abundant in cardiac and skeletal muscles, crosslinks actin filaments and contributes to Z-disc formation and membrane-cytoskeleton attachment. Here, we analyzed the interaction of a human filamin C fragment containing immunoglobulin-like domains 22-24 (FLNC22-24) with five small heat shock proteins (HspB1, HspB5, HspB6, HspB7, HspB8) and their α-crystallin domains. On size-exclusion chromatography, only HspB7 or its α-crystallin domain formed complexes with FLNC22-24. Despite similar isoelectric points of the small heat shock proteins analyzed, only HspB7 and its α-crystallin domain interacted with FLNC22-24 on native gel electrophoresis. Crosslinking with glutaraldehyde confirmed the formation of complexes between HspB7 (or its α-crystallin domain) and the filamin С fragment, inhibiting intersubunit FLNC crosslinking. These data are consistent with the structure modeling using Alphafold. Thus, the C-terminal fragment (immunoglobulin-like domains 22-24) of filamin C contains the site for HspB7 (or its α-crystallin domain) interaction, which competes with FLNC22-24 dimerization and its probable interaction with different target proteins.

Geranylgeranylacetone reduces cardiomyocyte stiffness and attenuates diastolic dysfunction in a rat model of cardiometabolic syndrome.

Titin-dependent stiffening of cardiomyocytes is a significant contributor to left ventricular (LV) diastolic dysfunction in heart failure with preserved LV ejection fraction (HFpEF). Small heat shock proteins (HSPs), such as HSPB5 and HSPB1, protect titin and administration of HSPB5 in vitro lowers cardiomyocyte stiffness in pressure-overload hypertrophy. In humans, oral treatment with geranylgeranylacetone (GGA) increases myocardial HSP expression, but the functional implications are unknown. Our objective was to investigate whether oral GGA treatment lowers cardiomyocyte stiffness and attenuates LV diastolic dysfunction in a rat model of the cardiometabolic syndrome. Twenty-one-week-old male lean (n = 10) and obese (n = 20) ZSF1 rats were studied, and obese rats were randomized to receive GGA (200 mg/kg/day) or vehicle by oral gavage for 4 weeks. Echocardiography and cardiac catheterization were performed before sacrifice at 25 weeks of age. Titin-based stiffness (Fpassive ) was determined by force measurements in relaxing solution with 100 nM [Ca2+ ] in permeabilized cardiomyocytes at sarcomere lengths (SL) ranging from 1.8 to 2.4 µm. In obese ZSF1 rats, GGA reduced isovolumic relaxation time of the LV without affecting blood pressure, EF or LV weight. In cardiomyocytes, GGA increased myofilament-bound HSPB5 and HSPB1 expression. Vehicle-treated obese rats exhibited higher cardiomyocyte stiffness at all SLs compared to lean rats, while GGA reduced stiffness at SL 2.0 µm. In obese ZSF1 rats, oral GGA treatment improves cardiomyocyte stiffness by increasing myofilament-bound HSPB1 and HSPB5. GGA could represent a potential novel therapy for the early stage of diastolic dysfunction in the cardiometabolic syndrome.

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.

Interplay between Nrf2 and αB-crystallin in the lens and heart of zebrafish under proteostatic stress.

A coordinated oxidative stress response, partly triggered by the transcription factor Nrf2, protects cells from the continual production of reactive oxygen species. Left unbuffered, reactive oxygen species can lead to protein aggregation that has been implicated in a spectrum of diseases such as cataract of the ocular lens and myopathy of the heart. While proteostasis is maintained by diverse families of heat shock proteins, the interplay between the oxidative and proteostatic stress responses in the lens and heart has not been investigated. Capitalizing on multiple zebrafish lines that have compromised function of Nrf2 and/or the two zebrafish small heat shock proteins αBa- and αBb-crystallin, we uncovered a transcriptional relationship that leads to a substantial increase in αBb-crystallin transcripts in the heart in response to compromised function of Nrf2. In the lens, the concomitant loss of function of Nrf2 and αBa-crystallin leads to upregulation of the cholesterol biosynthesis pathway, thus mitigating the phenotypic consequences of the αBa-crystallin knockout. By contrast, abrogation of Nrf2 function accentuates the penetrance of a heart edema phenotype characteristic of embryos of αB-crystallin knockout lines. Multiple molecular pathways, such as genes involved in extracellular interactions and implicated in cardiomyopathy, are revealed from transcriptome profiling, thus identifying novel targets for further investigation. Together, our transcriptome/phenotypic analysis establishes an intersection between oxidative stress and chaperone responses in the lens and heart.

The beauty and complexity of the small heat shock proteins: a report on the proceedings of the fourth workshop on small heat shock proteins.

The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.

CLPB3 is required for the removal of chloroplast protein aggregates and thermotolerance in Chlamydomonas.

In the cytosol of plant cells, heat-induced protein aggregates are resolved by the CASEIN LYTIC PROTEINASE/HEAT SHOCK PROTEIN 100 (CLP/HSP100) chaperone family member HSP101, which is essential for thermotolerance. For the chloroplast family member CLPB3 this is less clear, with controversial reports on its role in conferring thermotolerance. To shed light on this issue, we have characterized two clpb3 mutants in Chlamydomonas reinhardtii. We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TRIGGER FACTOR (TIG1) and the small heat shock proteins 22E/F (HSP22E/F) in vivo, and for conferring thermotolerance under heat stress. Although CLPB3 accumulation is similar to that of stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the PLASTID RIBOSOMAL PROTEIN L1 (PRPL1) and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that a reduction in chloroplast protein synthesis capacity and an increase in proteolytic capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast, but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to HSP22E/F, which accumulated largely near the thylakoid membranes. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system.

Small Heat Shock Protein (sHSP) Gene Family from Sweet Pepper (Capsicum annuum L.) Fruits: Involvement in Ripening and Modulation by Nitric Oxide (NO).

Small heat shock proteins (sHSPs) are usually upregulated in plants under diverse environmental stresses. These proteins have been suggested to function as molecular chaperones to safeguard other proteins from stress-induced damage. The ripening of pepper (Capsicum annuum L.) fruit involves important phenotypic, physiological, and biochemical changes, which have associated endogenous physiological nitro-oxidative stress, but they can also be significantly affected by environmental conditions, such as temperature. Based on the available pepper genome, a total of 41 sHSP genes were identified in this work, and their distributions in the 12 pepper chromosomes were determined. Among these genes, only 19 sHSP genes were found in the transcriptome (RNA-Seq) of sweet pepper fruits reported previously. This study aims to analyze how these 19 sHSP genes present in the transcriptome of sweet pepper fruits are modulated during ripening and after treatment of fruits with nitric oxide (NO) gas. The time-course expression analysis of these genes during fruit ripening showed that 6 genes were upregulated; another 7 genes were downregulated, whereas 6 genes were not significantly affected. Furthermore, NO treatment triggered the upregulation of 7 sHSP genes and the downregulation of 3 sHSP genes, whereas 9 genes were unchanged. These data indicate the diversification of sHSP genes in pepper plants and, considering that sHSPs are important in stress tolerance, the observed changes in sHSP expression support that pepper fruit ripening has an associated process of physiological nitro-oxidative stress, such as it was previously proposed.

α-Crystallin Domains of Five Human Small Heat Shock Proteins (sHsps) Differ in Dimer Stabilities and Ability to Incorporate Themselves into Oligomers of Full-Length sHsps.

The α-crystallin domain (ACD) is the hallmark of a diverse family of small heat shock proteins (sHsps). We investigated some of the ACD properties of five human sHsps as well as their interactions with different full-length sHsps. According to size-exclusion chromatography, at high concentrations, the ACDs of HspB1 (B1ACD), HspB5 (B5ACD) and HspB6 (B6ACD) formed dimers of different stabilities, which, upon dilution, dissociated to monomers to different degrees. Upon dilution, the B1ACD dimers possessed the highest stabilities, and those of B6ACD had the lowest. In striking contrast, the ACDs of HspB7 (B7ACD) and HspB8 (B8ACD) formed monomers in the same concentration range, which indicated the compromised stabilities of their dimer interfaces. B1ACD, B5ACD and B6ACD transiently interacted with full-length HspB1 and HspB5, which are known to form large oligomers, and modulated their oligomerization behavior. The small oligomers formed by the 3D mutant of HspB1 (mimicking phosphorylation at Ser15, Ser78 and Ser82) effectively interacted with B1ACD, B5ACD and B6ACD, incorporating these α-crystallin domains into their structures. The inherently dimeric full-length HspB6 readily formed heterooligomeric complexes with B1ACD and B5ACD. In sharp contrast to the abovementioned ACDs, B7ACD and B8ACD were unable to interact with full-length HspB1, the 3D mutant of HspB1, HspB5 or HspB6. Thus, their high sequence homology notwithstanding, B7ACD and B8ACD differ from the other three ACDs in their inability to form dimers and interact with the full-length small heat shock proteins. Having conservative primary structures and being apparently similar, the ACDs of the different sHsps differ in terms of their dimer stabilities, which can influence the heterooligomerization preferences of sHsps.

Differential Accumulation of sHSPs Isoforms during Desiccation of the Resurrection Plant Haberlea rhodopensis Friv. under Optimal and High Temperature.

Haberlea rhodopensis belongs to the small group of angiosperms that can survive desiccation to air-dry state and quickly restore their metabolism upon rehydration. In the present study, we investigated the accumulation of sHSPs and the extent of non-photochemical quenching during the downregulation of photosynthesis in H. rhodopensis leaves under desiccation at optimum (23 °C) and high temperature (38 °C). Desiccation of plants at 38 °C caused a stronger reduction in photosynthetic activity and corresponding enhancement in thermal energy dissipation. The accumulation of sHSPs was investigated by Western blot. While no expression of sHPSs was detected in the unstressed control sample, exposure of well-hydrated plants to high temperature induced an accumulation of sHSPs. Only a faint signal was observed at 50% RWC when dehydration was applied at 23 °C. Several cross-reacting polypeptide bands in the range of 16.5-19 kDa were observed in plants desiccated at high temperature. Two-dimensional electrophoresis and immunoblotting revealed the presence of several sHSPs with close molecular masses and pIs in the range of 5-8.0 that differed for each stage of treatment. At the latest stages of desiccation, fourteen different sHSPs could be distinguished, indicating that sHSPs might play a crucial role in H. rhodopensis under dehydration at high temperatures.

Disordered region encodes α-crystallin chaperone activity toward lens client γD-crystallin.

Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR ("Critical sequence") contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

HspB5 Chaperone Structure and Activity Are Modulated by Chemical-Scale Interactions in the ACD Dimer Interface.

Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that function as "holdases" and prevent protein aggregation due to changes in temperature, pH, or oxidation state. sHsps have a conserved α-crystallin domain (ACD), which forms the dimer building block, flanked by variable N- and C-terminal regions. sHsps populate various oligomeric states as a function of their sequestrase activity, and these dynamic structural features allow the proteins to interact with a plethora of cellular substrates. However, the molecular mechanisms of their dynamic conformational assembly and the interactions with various substrates remains unclear. Therefore, it is important to gain insight into the underlying physicochemical properties that influence sHsp structure in an effort to understand their mechanism(s) of action. We evaluated several disease-relevant mutations, D109A, F113Y, R116C, R120G, and R120C, in the ACD of HspB5 for changes to in vitro chaperone activity relative to that of wildtype. Structural characteristics were also evaluated by ANS fluorescence and CD spectroscopy. Our results indicated that mutation Y113F is an efficient holdase, while D109A and R120G, which are found in patients with myofibrillar myopathy and cataracts, respectively, exhibit a large reduction in holdase activity in a chaperone-like light-scattering assay, which indicated alterations in substrate-sHsp interactions. The extent of the reductions in chaperone activities are different among the mutants and specific to the substrate protein, suggesting that while sHsps are able to interact with many substrates, specific interactions provide selectivity for some substrates compared to others. This work is consistent with a model for chaperone activity where key electrostatic interactions in the sHsp dimer provide structural stability and influence both higher-order sHsp interactions and facilitate interactions with substrate proteins that define chaperone holdase activity.

The Common Bean Small Heat Shock Protein Nodulin 22 from Phaseolus vulgaris L. Assembles into Functional High-Molecular-Weight Oligomers.

Small heat shock proteins (sHsps) are present in all domains of life. These proteins are responsible for binding unfolded proteins to prevent their aggregation. sHsps form dynamic oligomers of different sizes and constitute transient reservoirs for folding competent proteins that are subsequently refolded by ATP-dependent chaperone systems. In plants, the sHsp family is rather diverse and has been associated with the ability of plants to survive diverse environmental stresses. Nodulin 22 (PvNod22) is an sHsp of the common bean (Phaseolus vulgaris L.) located in the endoplasmic reticulum. This protein is expressed in response to stress (heat or oxidative) or in plant roots during mycorrhizal and rhizobial symbiosis. In this work, we study its oligomeric state using a combination of in silico and experimental approaches. We found that recombinant PvNod22 was able to protect a target protein from heat unfolding in vitro. We also demonstrated that PvNod22 assembles into high-molecular-weight oligomers with diameters of ~15 nm under stress-free conditions. These oligomers can cluster together to form high-weight polydisperse agglomerates with temperature-dependent interactions; in contrast, the oligomers are stable regarding temperature.

Targeting small heat shock proteins to degrade aggregates as a potential strategy in neurodegenerative diseases.

Neurodegenerative diseases (NDs) are aging-related diseases that involve the death of neurons in the brain. Dysregulation of protein homeostasis leads to the production of toxic proteins or the formation of aggregates, which is the pathological basis of NDs. Small heat shock proteins (HSPB) is involved in the establishment of a protein quality control (PQC) system to maintain cellular homeostasis. HSPB can be secreted into the extracellular space and delivered by various routes, especially extracellular vehicles (EVs). HSPB plays an important role in influencing the aggregation phase of toxic proteins involved in heat shock transcription factor (HSF) regulation, oxidative stress, autophagy and apoptosis pathways. HSPB conferred neuroprotective effects by resisting toxic protein aggregation, reducing autophagy and reducing neuronal apoptosis. The HSPB treatment strategies, including targeted PQC system therapy and delivery of EVs-HSPB, can improve disease manifestations for NDs. This review aims to provide a comprehensive insight into the impact of HSPB in NDs and the feasibility of new technology to enhance HSPB expression and EVs-HSPB delivery for neurodegenerative disease.

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.

Explore the mechanism of incomplete Kawasaki disease and identify a novel biomarker by weighted gene co-expression network analysis.

Incomplete Kawasaki Disease is a complex disease that often occurs in infants and has substantial coronary artery damage. Its pathogenesis is unclear and lacks specific diagnostic markers. The purpose of our study is to research the mechanism of incomplete Kawasaki Disease use of bioinformatic methods and identify potential biomarkers. We performed weighted gene co-expression network analysis to analyze the data set GSE68004 and identified modules and genes which were correlated with the disease. Through functional annotation and enrichment analysis, we determined the biological function and signal pathway of these genes. We further used lasso regression and ROC curve to screen genes and determined that the final candidate gene was HSPB11and hsa-miR-155-5p that regulates its expression. Finally, we validated the screened gene using an independent dataset and construct a TF-miRNA network. Through the relationships of TFs and hsa-miR-155-5p, we found is hsa-miR-155-5p closely related to hypoxia-related transcription factors, which may be a new direction in the research of Kawasaki disease.

Role of Small Heat Shock Proteins in the Remodeling of Actin Microfilaments.

Small heat shock proteins (sHsps) play an important role in the maintenance of proteome stability and, particularly, in stabilization of the cytoskeleton and cell contractile apparatus. Cell exposure to different types of stress is accompanied by the translocation of sHsps onto actin filaments; therefore, it is commonly believed that the sHsps are true actin-binding proteins. Investigations of last years have shown that this assumption is incorrect. Stress-induced translocation of sHsp to actin filaments is not the result of direct interaction of these proteins with intact actin, but results from the chaperone-like activity of sHsps and their interaction with various actin-binding proteins. HspB1 and HspB5 interact with giant elastic proteins titin and filamin thus providing an integrity of the contractile apparatus and its proper localization in the cell. HspB6 binds to the universal adapter protein 14-3-3 and only indirectly affects the structure of actin filament. HspB7 interacts with filamin C and controls actin filament assembly. HspB8 forms tight complex with the universal regulatory and adapter protein Bag3 and participates in the chaperone-assisted selective autophagy (CASA) of actin-binding proteins (e.g., filamin), as well as in the actin-depending processes taking place in mitoses. Hence, the mechanisms of sHsp participation in the maintenance of the contractile apparatus and cytoskeleton are much more complicated and diverse than it has been postulated earlier and are not limited to direct interactions of sHsps with actin. The old hypothesis on the direct binding of sHsps to intact actin should be revised and further detailed investigation on the sHsp interaction with minor proteins participating in the formation and remodeling of actin filaments is required.

Is the small heat shock protein HSPB7 (cvHsp) a genuine actin-binding protein?

It is postulated that the small heat shock proteins directly interact with actin, affect formation and stabilize actin filaments. To verify this suggestion, we have analyzed interaction of recombinant human small heat shock protein HspB7 with skeletal muscle actin. In blot overlay HspB7 binds both G- and F-actin. The sites of interaction are located in the C-terminal large core domain of actin. In the course of ultracentrifugation F-actin and F-actin/tropomyosin complexes were pelleted and trapped HspB7. However, HspB7 pelleting was nonspecific and saturation was not achieved even at very high HspB7 concentration. HspB7 was unable to retard or prevent heat-induced F-actin aggregation. Native gel electrophoresis and chemical crosslinking failed to detect interaction of G-actin with HspB7, although both these methods clearly demonstrated formation of complexes formed by G-actin with DNAse I and cofilin-2. It is concluded that HspB7 is not a genuine actin-binding protein and its effect on actin filaments seems to be determined by interaction of HspB7 with minor regulatory proteins of actin filaments.