A model for COVID-19-induced dysregulation of ACE2 shedding by ADAM17.

The angiotensin Converting Enzyme 2 (ACE2) receptor is a key component of the renin-angiotensin-aldesterone system (RAAS) that mediates numerous effects in the cardiovascular system. It is also the cellular point of contact for the coronavirus spike protein. Cleavage of the receptor is both important to its physiological function as well as being necessary for cell entry by the virus. Shedding of ACE2 by the metalloprotease ADAM17 releases a catalytically active soluble form of ACE2, but cleavage by the serine protease TMPRSS2 is necessary for virion internalization. Complicating the issue is the observation that circulating ACE2 can also bind to the virus effectively blocking attachment to the membrane-bound receptor. This work investigates the possibility that the inflammatory response to coronavirus infection can abrogate shedding by ADAM17, thereby favoring cleavage by TMPRSS2 and thus cell entry by the virion.

Physico-chemical properties of two point mutants of small heat shock protein HspB6 (Hsp20) with abrogated cardioprotection.

Physico-chemical properties of HspB6 S10F and P20L mutants with abrogated cardioprotective activity and associated with different forms of cardiomyopathy were analyzed. Under normal conditions both the wild-type HspB6 and its mutants formed small size oligomers (dimers) with apparent molecular weight of 50-60 kDa. Under crowding conditions (0.5 M trimethylamine N-oxide, TMAO) the wild-type HspB6 remained predominantly dimeric or formed small molecular weight complexes, whereas both mutants tended to form high molecular weight complexes. Catalytic subunit of cAMP-dependent protein kinase phosphorylated the wild-type HspB6 and its S10F mutant with comparable rate. The rate of P20L mutant phosphorylation was higher than that of the wild-type HspB6. S10F and P20L mutations did not affect interaction of phosphorylated HspB6 with universal adapter proteins 14-3-3. The wild-type HspB6 was resistant to heat-induced denaturation and aggregation, whereas both its mutants were denatured and started to aggregate at temperature much lower than its wild-type counterpart. Titration with fluorescent probe bis-ANS was accompanied by larger increase of fluorescence in the case of both mutants than in the case of the wild-type HspB6. Both mutants possessed higher chaperone-like activity than the wild-type protein. It is concluded that both S10F and P20L mutations are accompanied by increase of hydrophobicity of the very N-terminal region of HspB6 leading to increased aggregation at elevated temperature, formation of large complexes under crowding conditions and increased chaperone-like activity measured in vitro. Increased hydrophobicity and self-association can affect substrate specificity and interaction with certain target proteins thus leading to decrease or complete abrogation of cardioprotective activity.

Effect of Arginine on Chaperone-Like Activity of HspB6 and Monomeric 14-3-3ζ.

The effect of protein chaperones HspB6 and the monomeric form of the protein 14-3-3ζ (14-3-3ζm) on a test system based on thermal aggregation of UV-irradiated glycogen phosphorylase b (UV-Phb) at 37 °C and a constant ionic strength (0.15 M) was studied using dynamic light scattering. A significant increase in the anti-aggregation activity of HspB6 and 14-3-3ζm was demonstrated in the presence of 0.1 M arginine (Arg). To compare the effects of these chaperones on UV-Phb aggregation, the values of initial stoichiometry of the chaperone-target protein complex (S0) were used. The analysis of the S0 values shows that in the presence of Arg fewer chaperone subunits are needed to completely prevent aggregation of the UV-Phb subunit. The changes in the structures of HspB6 and 14-3-3ζm induced by binding of Arg were evaluated by the fluorescence spectroscopy and differential scanning calorimetry. It was suggested that Arg caused conformational changes in chaperone molecules, which led to a decrease in the thermal stability of protein chaperones and their destabilization.

The Role of the Arginine in the Conserved N-Terminal Domain RLFDQxFG Motif of Human Small Heat Shock Proteins HspB1, HspB4, HspB5, HspB6, and HspB8.

Although the N-terminal domain of vertebrate small heat shock proteins (sHsp) is poorly conserved, it contains a core motif preserved in many members of the sHsp family. The role of this RLFDQxFG motif remains elusive. We analyzed the specific role of the first arginine residue of this conserved octet sequence in five human sHsps (HspB1, HspB4, HspB5, HspB6, and HspB8). Substitution of this arginine with an alanine induced changes in thermal stability and/or intrinsic fluorescence of the related HspB1 and HspB8, but yielded only modest changes in the same biophysical properties of HspB4, HspB5, and HspB6 which together belong to another clade of vertebrate sHsps. Removal of the positively charged Arg side chain resulted in destabilization of the large oligomers of HspB1 and formation of smaller size oligomers of HspB5. The mutation induced only minor changes in the structure of HspB4 and HspB6. In contrast, the mutation in HspB8 was accompanied by shifting the equilibrium from dimers towards the formation of larger oligomers. We conclude that the RLFDQxFG motif plays distinct roles in the structure of several sHsp orthologs. This role correlates with the evolutionary relationship of the respective sHsps, but ultimately, it reflects the sequence context of this motif.

Characterization of human small heat shock protein HSPB1 α-crystallin domain localized mutants associated with hereditary motor neuron diseases.

Congenital mutations in human small heat shock protein HSPB1 (HSP27) have been linked to Charcot-Marie-Tooth disease, a commonly occurring peripheral neuropathy. Understanding the molecular mechanism of such mutations is indispensable towards developing future therapies for this currently incurable disorder. Here we describe the physico-chemical properties of the autosomal dominant HSPB1 mutants R127W, S135F and R136W. Despite having a nominal effect on thermal stability, the three mutations induce dramatic changes to quaternary structure. At high concentrations or under crowding conditions, the mutants form assemblies that are approximately two times larger than those formed by the wild-type protein. At low concentrations, the mutants have a higher propensity to dissociate into small oligomers, while the dissociation of R127W and R135F mutants is enhanced by MAPKAP kinase-2 mediated phosphorylation. Specific differences are observed in the ability to form hetero-oligomers with the homologue HSPB6 (HSP20). For wild-type HSPB1 this only occurs at or above physiological temperature, whereas the R127W and S135F mutants form hetero-oligomers with HSPB6 at 4 °C, and the R136W mutant fails to form hetero-oligomers. Combined, the results suggest that the disease-related mutations of HSPB1 modify its self-assembly and interaction with partner proteins thus affecting normal functioning of HSPB1 in the cell.

Study of HSPB6: Insights into the Properties of the Multifunctional Protective Agent.

HSPB6(Heat shock protein B6), is also referred to as P20/HSP20. Unlike other many other members of sHSP(small Heat shock protein) family, which tend to form high-molecular-mass oligomers, in solution, human HSPB6 only forms dimers. However, it still exhibits chaperon-like activity comparable with that of HSPB5. It is expressed ubiquitously, with high and constitutive expression in muscular tissues. sHSPs characteristically function as molecular chaperones and HSPB6 also has a molecular chaperone activity. HSPB6 is up-regulated in response to diverse cellular stress or damage and protect cells from otherwise lethal conditions. HSPB6 is widely recognized as a principle mediator of cardioprotective signaling and recent studies have unraveled the protective role of HSPB6 in disease or injury to the central nervous system. Moreover, accumulating evidence has implicated HSPB6 as a key mediator of diverse vital physiological processes, such as smooth muscle relaxation, platelet aggregation. The versatility of HSPB6 can be explained by its direct involvement in regulating different client proteins and its ability to form heterooligomer with other sHSPs, which seems to be dependent on HSPB6 phosphorylation. This review focuses on the properties including expression and regulation pattern, phosphorylation, chaperon activity, multiple cellular targets of HSPB6, as well as its possible role in physical and pathological conditions.

Characterization and expression analysis of a new small heat shock protein Hsp20.4 from Eimeria tenella.

Small heat shock proteins (sHsps) are ubiquitous and diverse molecular chaperones. Found in almost all organisms, they regulate protein refolding and protect cells from stress. Until now, no sHsp has been characterized in Eimeria tenella. In this study, the novel EtsHsp20.4 gene was cloned from E. tenella by rapid amplification of cDNA ends based on a previously identified expressed sequence tag. The full-length cDNA was 1019bp in length and contained an open reading frame of 558bp that encoded a 185-amino acid polypeptide with a calculated molecular weight of 20.4 kDa. The EtsHsp20.4 protein contained a distinct HSP20/alpha-crystallin domain that is the key determinant of their function as molecular chaperones and belongs to the HSP20 protein family. EtsHsp20.4 mRNA levels were higher in sporulated oocysts than in sporozoites or second-generation merozoites by real-time quantitative PCR, the transcription of EtsHsp20.4 was barely detectable in unsporulated oocysts. Immunolocalization with EtsHsp20.4 antibody showed that EtsHsp20.4 was mainly located on the surface of sporozoites, first-generation merozoites and second-generation merozoites. Following the development of parasites in DF-1 cells, EtsHsp20.4 protein was uniformly dispersed in trophozoites, immature schizonts, and mature schizonts. Malate dehydrogenase thermal aggregation assays indicated that recombinant EtsHsp20.4 had molecular chaperone activity in vitro. These results suggested that EtsHsp20.4 might be involved in sporulation in external environments and intracellular growth of the parasite in the host.

Methylglyoxal and Small Heat Shock Proteins.

Methylglyoxal is a highly reactive dicarbonyl compound formed during glucose metabolism and able to modify phospholipids, nucleic acids, and proteins belonging to the so-called dicarbonyl proteome. Small heat shock proteins participating in protection of the cell against different unfavorable conditions can be modified by methylglyoxal. The probability of methylglyoxal modification is increased in the case of distortion of glucose metabolism (diabetes), in the case of utilization of glycolysis as the main source of energy (malignancy), and/or at low rate of modified protein turnover. We have analyzed data on modification of small heat shock protein HspB1 in different tumors and under distortion of carbohydrate metabolism. Data on the effect of methylglyoxal modification on stability, chaperone-like activity, and antiapoptotic activity of HspB1 were analyzed. We discuss data on methylglyoxal modifications of lens α-crystallins. The mutual dependence and mutual effects of methylglyoxal modification and other posttranslational modifications of lens crystallins are analyzed. We conclude that although there is no doubt that the small heat shock proteins undergo methylglyoxal modification, the physiological significance of this process remains enigmatic, and new experimental approaches should be developed for understanding how this type of modification affects functioning of small heat shock proteins in the cell.

Specific sequences in the N-terminal domain of human small heat-shock protein HSPB6 dictate preferential hetero-oligomerization with the orthologue HSPB1.

Small heat-shock proteins (sHSPs) are a conserved group of molecular chaperones with important roles in cellular proteostasis. Although sHSPs are characterized by their small monomeric weight, they typically assemble into large polydisperse oligomers that vary in both size and shape but are principally composed of dimeric building blocks. These assemblies can include different sHSP orthologues, creating additional complexity that may affect chaperone activity. However, the structural and functional properties of such hetero-oligomers are poorly understood. We became interested in hetero-oligomer formation between human heat-shock protein family B (small) member 1 (HSPB1) and HSPB6, which are both highly expressed in skeletal muscle. When mixed in vitro, these two sHSPs form a polydisperse oligomer array composed solely of heterodimers, suggesting preferential association that is determined at the monomer level. Previously, we have shown that the sHSP N-terminal domains (NTDs), which have a high degree of intrinsic disorder, are essential for the biased formation. Here we employed iterative deletion mapping to elucidate how the NTD of HSPB6 influences its preferential association with HSPB1 and show that this region has multiple roles in this process. First, the highly conserved motif RLFDQXFG is necessary for subunit exchange among oligomers. Second, a site ∼20 residues downstream of this motif determines the size of the resultant hetero-oligomers. Third, a region unique to HSPB6 dictates the preferential formation of heterodimers. In conclusion, the disordered NTD of HSPB6 helps regulate the size and stability of hetero-oligomeric complexes, indicating that terminal sHSP regions define the assembly properties of these proteins.

Immunodominant protein MIP_05962 from Mycobacterium indicus pranii displays chaperone activity.

Tuberculosis, a contagious disease of infectious origin is currently a major cause of deaths worldwide. Mycobacterium indicus pranii (MIP), a saprophytic nonpathogen and a potent immunomodulator is currently being investigated as an intervention against tuberculosis along with many other diseases with positive outcome. The apparent paradox of multiple chaperones in mycobacterial species and enigma about the cellular functions of the client proteins of these chaperones need to be explored. Chaperones are the known immunomodulators; thus, there is need to exploit the proteome of MIP for identification and characterization of putative chaperones. One of the immunogenic proteins, MIP_05962 is a member of heat shock protein (HSP) 20 family due to the presence of α-crystallin domain, and has amino acid similarity with Mycobacterium lepraeHSP18 protein. The diverse functions of M. lepraeHSP18 in stress conditions implicate MIP_05962 as an important protein that needs to be explored. Biophysical and biochemical characterization of the said protein proved it to be a chaperone. The observations of aggregation prevention and refolding of substrate proteins in the presence of MIP_05962 along with interaction with non-native proteins, surface hydrophobicity, formation of large oligomers, in-vivo thermal rescue of Escherichia coli expressing MIP_05962, enhancing solubility of insoluble protein maltodextrin glucosidase (MalZ) under in-vivo conditions, and thermal stability and reversibility confirmed MIP_05962 as a molecular chaperone.

Structural Basis for the Interaction of a Human Small Heat Shock Protein with the 14-3-3 Universal Signaling Regulator.

By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein, HSPB6, within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering, and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants.

The preferential heterodimerization of human small heat shock proteins HSPB1 and HSPB6 is dictated by the N-terminal domain.

Small heat shock proteins are ATP-independent molecular chaperones. Their function is to bind partially unfolded proteins under stress conditions. In vivo, members of this chaperone family are known to preferentially assemble together forming large, polydisperse heterooligomers. The exact molecular mechanisms that drive specific heteroassociation are currently unknown. Here we study the oligomers formed between human HSPB1 and HSPB6. Using small-angle X-ray scattering we could characterize two distinct heterooligomeric species present in solution. By employing native mass spectrometry we show that such assemblies are formed purely from heterodimeric building blocks, in line with earlier cross-linking studies. Crucially, a detailed analysis of truncation variants reveals that the preferential association between these two sHSPs is solely mediated by their disordered N-terminal domains.

Molecular cloning and expression analysis of heat shock protein 20 (HSP20) from the pearl oyster Pinctada martensii.

Small heat shock proteins (HSPs) are molecular chaperones with ATP-independent properties. They are involved in a variety of physiological and stress processes. In this study, the full-length HSP 20 (HSP20) from Pinctada martensii, designated as PmHSP20, was obtained from hemocytes using rapid amplification of cDNA ends technology. The PmHSP20 cDNA was 952 bp in length, containing an open reading frame of 534 bp that encoded 177-amino acid residues, with an isoelectric point of 5.86 and molecular weight of 20.24 kDa. The sequence of this deduced polypeptide contained typical structure and function domains conserved in the HSP20 family, providing evidence that PmHSP20 belongs to the HSP20 family. The PmHSP20 mRNA expression levels were detected in various tissues of P. martensii and in hemocytes after challenges with the bacteria Vibrio harveyi and lipopolysaccharide (LPS) using quantitative real-time polymerase chain reaction amplification. The results indicated that PmHSP20 is constitutively expressed in all tissues tested and might be involved in the immune response. The upregulation of PmHSP20 after V. harveyi and LPS challenge suggests that PmHSP20 plays an important role in anti-bacterial immunity. Studies on PmHSP20 are a valuable resource to further explore the immune system in pearl oysters and might enhance our knowledge of molluscan innate immunity.

Expression and localization of the Xenopus laevis small heat shock protein, HSPB6 (HSP20), in A6 kidney epithelial cells.

Small heat shock proteins (sHSPs) are molecular chaperones that bind to unfolded protein, inhibit the formation of toxic aggregates and facilitate their refolding and/or degradation. Previously, the only sHSPs that have been studied in detail in the model frog system, Xenopus laevis, were members of the HSP30 family and HSPB1 (HSP27). We now report the analysis of X. laevis HSPB6, an ortholog of mammalian HSPB6. X. laevis HSPB6 cDNA encodes a 168 aa protein that contains an α-crystallin domain, a polar C-terminal extension and some possible phosphorylation sites. X. laevis HSPB6 shares 94% identity with a X. tropicalis HSPB6, 65% with turtle, 59% with humans, 49% with zebrafish and only 50% and 43% with X. laevis HSPB1 and HSP30C, respectively. Phylogenetic analysis revealed that X. laevis HSPB6 grouped more closely with mammalian and reptilian HSPB6s than with fish HSPB6. X. laevis recombinant HSPB6 displayed molecular chaperone properties since it had the ability to inhibit heat-induced aggregation of citrate synthase. Immunoblot analysis determined that HSPB6 was present constitutively in kidney epithelial cells and that heat shock treatment did not upregulate HSPB6 levels. While treatment with the proteasomal inhibitor, MG132, resulted in a 2-fold increase in HSPB6 levels, exposure to cadmium chloride produced a slight increase in HSPB6. These findings were in contrast to HSP70, which was enhanced in response to all three stressors. Finally, immunocytochemical analysis revealed that HSPB6 was present in the cytoplasm in the perinuclear region with some in the nucleus.

Effect of methylglyoxal modification on the structure and properties of human small heat shock protein HspB6 (Hsp20).

Human small heat shock protein HspB6 (Hsp20) was modified by metabolic α-dicarbonyl compound methylglyoxal (MGO). At low MGO/HspB6 molar ratio, Arg13, Arg14, Arg27, and Arg102 were the primary sites of MGO modification. At high MGO/HspB6 ratio, practically, all Arg and Lys residues of HspB6 were modified. Both mild and extensive MGO modification decreased susceptibility of HspB6 to trypsinolysis and prevented its heat-induced aggregation. Modification by MGO was accompanied by formation of small quantities of chemically crosslinked dimers and did not dramatically affect quaternary structure of HspB6. Mild modification by MGO did not affect whereas extensive modification decreased interaction of HspB6 with HspB1. Phosphorylation of HspB6 by cyclic adenosine monophosphate (cAMP)-dependent protein kinase was inhibited after mild modification and completely prevented after extensive modification by MGO. Chaperone-like activity of HspB6 measured with subfragment 1 of skeletal myosin was enhanced after MGO modifications. It is concluded that Arg residues located in the N-terminal domain of HspB6 are easily accessible to MGO modification and that even mild modification by MGO affects susceptibility to trypsinolysis, phosphorylation by cAMP-dependent protein kinase, and chaperone-like activity of HspB6.

Characterization of Mutants of Human Small Heat Shock Protein HspB1 Carrying Replacements in the N-Terminal Domain and Associated with Hereditary Motor Neuron Diseases.

Physico-chemical properties of the mutations G34R, P39L and E41K in the N-terminal domain of human heat shock protein B1 (HspB1), which have been associated with hereditary motor neuron neuropathy, were analyzed. Heat-induced aggregation of all mutants started at lower temperatures than for the wild type protein. All mutations decreased susceptibility of the N- and C-terminal parts of HspB1 to chymotrypsinolysis. All mutants formed stable homooligomers with a slightly larger apparent molecular weight compared to the wild type protein. All mutations analyzed decreased or completely prevented phosphorylation-induced dissociation of HspB1 oligomers. When mixed with HspB6 and heated, all mutants yielded heterooligomers with apparent molecular weights close to ~400 kDa. Finally, the three HspB1 mutants possessed lower chaperone-like activity towards model substrates (lysozyme, malate dehydrogenase and insulin) compared to the wild type protein, conversely the environmental probe bis-ANS yielded higher fluorescence with the mutants than with the wild type protein. Thus, in vitro the analyzed N-terminal mutations increase stability of large HspB1 homooligomers, prevent their phosphorylation-dependent dissociation, modulate their interaction with HspB6 and decrease their chaperoning capacity, preventing normal functioning of HspB1.

[Effect of heat shock on cells of phytopathogenic mycoplasma Acholeplasma laidlawii PG-8A].

Heat shock caused a more active formation of the "dormant" forms (minibodies), as well as increased production of extracellular membrane vesicles by Acholeplasma laidlawii PG-8A cells. Raise of the amount of the minibodies that have increased resistance to biogenic and abiogenic stress factors and pathogenicity may lead to more successful persistence of mycoplasmas in their hosts. Increased production of the extracellular membrane vesicles containing virulence factors by Acholeplasma laidlawii cells during stress may be an additional burden for the infected organism. It has been recently revealed that the vesicles of A. laidlawii contain appreciable quantities of small heat shock protein IbpA (Hsp20). In this paper, using immune-electron microscopy, have shown that at elevated temperature IbpA is associated with A. laidlawii minibodies. Perhaps, IbpA contributes to increased resistance and pathogenicity of the minibodies, keeping their proteins and polypeptides, including protein virulence factors in the folding-competent state.

Crystal structure and function of an unusual dimeric Hsp20.1 provide insight into the thermal protection mechanism of small heat shock proteins.

Small heat shock proteins (sHSPs) are ubiquitous chaperones that play a vital role in protein homeostasis. sHSPs are characterized by oligomeric architectures and dynamic exchange of subunits. The flexible oligomeric assembling associating with function remains poorly understood. Based on the structural data, it is certainly agreed that two dimerization models depend on the presence or absence of a ß6 strand to differentiate nonmetazoan sHSPs from metazoan sHSPs. Here, we report the Sulfolobus solfataricus Hsp20.1 ACD dimer structure, which shows a distinct dimeric interface. We observed that, in the absence of ß6, Hsp20.1 dimer does not depend on ß7 strand for forming dimer interface as metazoan sHSPs, nor dissociates to monomers. This is in contrast to other published sHSPs. Our structure reveals a variable, highly polar dimer interface that has advantages for rapid subunits exchange and substrate binding. Remarkably, we find that the C-terminal truncation variant has chaperone activity comparable to that of wild-type despite lack of the oligomer structure. Our further study indicates that the N-terminal region is essential for the oligomer and dimer binding to the target protein. Together, the structure and function of Hsp20.1 give more insight into the thermal protection mechanism of sHSPs.

Quaternary structure of human small heat shock protein HSPB6 (Hsp20) in crowded media modeled by trimethylamine N-oxide (TMAO): Effect of protein phosphorylation.

Effect of trimethylamine N-oxide (TMAO), well-known osmolyte, widely used to imitate crowded intracellular conditions, on the quaternary structure of recombinant human small heat shock protein HspB6 (Hsp20) was analyzed by means of size-exclusion chromatography, chemical crosslinking and analytical ultracentrifugation. Consistent with previous reports, in the absence of TMAO unphosphorylated, pseudophosphorylated (S16D mutant) and phosphorylated HspB6 form only small oligomers (presumably dimers). Addition of TMAO to unphosphorylated HspB6 leads to formation of different large oligomers being in equilibrium with dimers. Pseudophosphorylation (S16D mutation) or phosphorylation partially or completely prevent TMAO-induced oligomerization of HspB6. Pseudophosphorylation affects bis-ANS binding suggesting decreased hydrophobicity of HspB6. According to size-exclusion chromatography, TMAO-induced changes of HspB6 oligomerization result in its altered interaction with HspB1 and this effect can be reversed by HspB6 phosphorylation. It is concluded that under conditions of molecular crowding, characteristic for intracellular environment, HspB6 undergoes reversible changes of its oligomeric state which can affect its physiologically important properties and can be delicately regulated by phosphorylation.