Human HspB1, HspB3, HspB5 and HspB8: Shaping these disease factors during vertebrate evolution.

Small heat shock proteins (sHSPs) emerged early in evolution and occur in all domains of life and nearly in all species, including humans. Mutations in four sHSPs (HspB1, HspB3, HspB5, HspB8) are associated with neuromuscular disorders. The aim of this study is to investigate the evolutionary forces shaping these sHSPs during vertebrate evolution. We performed comparative evolutionary analyses on a set of orthologous sHSP sequences, based on the ratio of non-synonymous: synonymous substitution rates for each codon. We found that these sHSPs had been historically exposed to different degrees of purifying selection, decreasing in this order: HspB8 > HspB1, HspB5 > HspB3. Within each sHSP, regions with different degrees of purifying selection can be discerned, resulting in characteristic selective pressure profiles. The conserved α-crystallin domains were exposed to the most stringent purifying selection compared to the flanking regions, supporting a 'dimorphic pattern' of evolution. Thus, during vertebrate evolution the different sequence partitions were exposed to different and measurable degrees of selective pressures. Among the disease-associated mutations, most are missense mutations primarily in HspB1 and to a lesser extent in the other sHSPs. Our data provide an explanation for this disparate incidence. Contrary to the expectation, most missense mutations cause dominant disease phenotypes. Theoretical considerations support a connection between the historic exposure of these sHSP genes to a high degree of purifying selection and the unusual prevalence of genetic dominance of the associated disease phenotypes. Our study puts the genetics of inheritable sHSP-borne diseases into the context of vertebrate evolution.

Sulfatase 2 Is Associated with Steroid Resistance in Childhood Nephrotic Syndrome.

Glucocorticoid (GC) resistance complicates the treatment of ~10-20% of children with nephrotic syndrome (NS), yet the molecular basis for resistance remains unclear. We used RNAseq analysis and in silico algorithm-based approaches on peripheral blood leukocytes from 12 children both at initial NS presentation and after ~7 weeks of GC therapy to identify a 12-gene panel able to differentiate steroid resistant NS (SRNS) from steroid-sensitive NS (SSNS). Among this panel, subsequent validation and analyses of one biologically relevant candidate, sulfatase 2 (SULF2), in up to a total of 66 children, revealed that both SULF2 leukocyte expression and plasma arylsulfatase activity Post/Pre therapy ratios were greater in SSNS vs. SRNS. However, neither plasma SULF2 endosulfatase activity (measured by VEGF binding activity) nor plasma VEGF levels, distinguished SSNS from SRNS, despite VEGF's reported role as a downstream mediator of SULF2's effects in glomeruli. Experimental studies of NS-related injury in both rat glomeruli and cultured podocytes also revealed decreased SULF2 expression, which were partially reversible by GC treatment of podocytes. These findings together suggest that SULF2 levels and activity are associated with GC resistance in NS, and that SULF2 may play a protective role in NS via the modulation of downstream mediators distinct from VEGF.

Small heat shock protein speciation: novel non-canonical 44 kDa HspB5-related protein species in rat and human tissues.

When analyzing small stress proteins of rat and human tissues by electrophoretic methods followed by western blotting, and using the anti-HspB1/anti-HspB5 antibody clone 8A7, we unexpectedly found a protein with a molecular mass of ~44 kDa. On two-dimensional gels, this protein resolved into four distinct species. Electrophoretic and immunological evidence suggests that this 44 kDa protein is a derivative of HspB5, most likely a covalently linked HspB5 dimer. This HspB5-like 44 kDa protein (HspB5L-P44) is particularly abundant in rat heart, brain, and renal cortex and glomeruli. HspB5L-P44 was also found in human brains, including those from patients with Alexander disease, a condition distinguished by cerebral accumulation of HspB5. Gray matter of such a patient contained an elevated amount of HspB5L-P44. A spatial model of structurally ordered dimeric HspB5 α-crystallin domains reveals the exposed and adjacent position of the two peptide segments homologous to the HspB1-derived 8A7 antigen determinant peptide (epitope). This explains the observed extraordinary high avidity of the 8A7 antibody towards HspB5L-P44, as opposed to commonly used HspB5-specific antibodies which recognize other epitopes. This scenario also explains the remarkable fact that no previous study reported the existence of HspB5L-P44 species. Exposure of rat endothelial cells to UV light, an oxidative stress condition, temporarily increased HspB5L-P44, suggesting physiological regulation of the dimerization. The existence of HspB5L-P44 supports the protein speciation discourse and fits to the concept of the protein code, according to which the expression of a given gene is reflected only by the complete set of the derived protein species.

Neuropathy- and myopathy-associated mutations in human small heat shock proteins: Characteristics and evolutionary history of the mutation sites.

Mutations in four of the ten human small heat shock proteins (sHSP) are associated with various forms of motor neuropathies and myopathies. In HspB1, HspB3, and HspB8 all known mutations cause motor neuropathies, whereas in HspB5 they cause myopathies. Several features are common to the majority of these mutations: (i) they are missense mutations, (ii) most associated disease phenotypes exhibit a dominant inheritance pattern and late disease onset, (iii) in the primary protein sequences, the sites of most mutations are located in the conserved α-crystallin domain and the variable C-terminal extensions, and (iv) most human mutation sites are highly conserved among the vertebrate orthologs and have been historically exposed to significant purifying selection. In contrast, a minor fraction of these mutations deviate from these rules: they are (i) frame shifting, nonsense, or elongation mutations, (ii) associated with recessive or early onset disease phenotypes, (iii) positioned in the N-terminal domain of the proteins, and (iv) less conserved among the vertebrates and were historically not subject to a strong selective pressure. In several vertebrate sHSPs (including primate sHSPs), homologous sites differ from the human sequence and occasionally even encode the same amino acid residues that cause the disease in humans. Apparently, a number of these mutations sites are not crucial for the protein function in single species or entire taxa, and single species even seem to have adopted mechanisms that compensate for potentially adverse effects of 'mutant-like' sHSPs. The disease-associated dominant sHSP missense mutations have a number of cellular consequences that are consistent with gain-of-function mechanisms of genetic dominance: dominant-negative effects, the formation of cytotoxic amyloid protein oligomers and precipitates, disruption of cytoskeletal networks, and increased downstream enzymatic activities. Future therapeutic concepts should aim for reducing these adverse effects of mutant sHSPs in patients. Indeed, initial experimental results are encouraging.

Glutathione reductase is essential for host defense against bacterial infection.

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, a major cellular antioxidant. We have recently shown that Gsr is essential for host defense against the gram-negative bacteria Escherichia coli in a mouse model of sepsis. Although we have demonstrated that Gsr is required for sustaining the oxidative burst and the development of neutrophil extracellular traps, the role of Gsr in other phagocytic functions remains unclear. It is also unclear whether Gsr-deficient mice exhibit host defense defects against gram-positive bacteria. In this study, we characterized the effects of Gsr deficiency on the innate immune responses to a gram-positive bacterium, group B Streptococcus, and to the gram-negative bacterial cell wall component lipopolysaccharide (LPS). We found that, like E. coli, group B Streptococcus resulted in a substantially more robust cytokine response and a markedly higher morbidity and mortality in Gsr-deficient mice than in wild-type mice. The increased morbidity and mortality were associated with greater bacterial burden in the Gsr-deficient mice. Interestingly, Gsr-deficient mice did not exhibit a greater sensitivity to LPS than did wild-type mice. Analysis of the neutrophils of Gsr-deficient mice revealed impaired phagocytosis. In response to thioglycollate stimulation, Gsr-deficient mice mobilized far fewer phagocytes, including neutrophils, macrophages, and eosinophils, into their peritoneal cavities than did wild-type mice. The defective phagocyte mobilization is associated with profound oxidation and aggregation of ascitic proteins, particularly albumin. Our results indicate that the oxidative defense mechanism mediated by Gsr is required for an effective innate immune response against bacteria, probably by preventing phagocyte dysfunction due to oxidative damage.

Crucial roles of the protein kinases MK2 and MK3 in a mouse model of glomerulonephritis.

Elevated mitogen-activated protein kinase p38 (p38 MAPK) signaling has been implicated in various experimental and human glomerulopathies, and its inhibition has proven beneficial in animal models of these diseases. p38 MAPK signaling is partially mediated through MK2 and MK3, two phylogenetically related protein kinases that are its direct substrates. The current study was designed to determine the specific roles of MK2 and MK3 in a mouse model of acute proliferative glomerulonephritis, using mice with disrupted MK2 and/or MK3 genes. We found that the absence of MK3 alone worsened the disease course and increased mortality slightly compared to wild-type mice, whereas the absence of MK2 alone exhibited no significant effect. However, in an MK3-free background, the disease course depended on the presence of MK2 in a gene dosage-dependent manner, with double knock-out mice being most susceptible to disease induction. Histological and renal functional analyses confirmed kidney damage following disease induction. Because the renal stress response plays a crucial role in kidney physiology and disease, we analyzed the stress response pattern in this disease model. We found that renal cortices of diseased mice exhibited a pronounced and specific pattern of expression and/or phosphorylation of stress proteins and other indicators of the stress response (HSPB1, HSPB6, HSPB8, CHOP, eIF2α), partially in a MK2/MK3 genotype-specific manner, and without induction of a general stress response. Similarly, the expression and activation patterns of other protein kinases downstream of p38 MAPK (MNK1, MSK1) depended partially on the MK2/MK3 genotype in this disease model. In conclusion, MK2 and MK3 together play crucial roles in the regulation of the renal stress response and in the development of glomerulonephritis, which can potentially be exploited to develop novel therapeutic approaches to treat glomerular disease.

Childhood nephrotic syndrome--current and future therapies.

The introduction of corticosteroids more than 50 years ago dramatically improved the prognosis of children with nephrotic syndrome. Corticosteroids remain the standard initial treatment for children with this disease, but a considerable proportion of patients do not respond and are therefore at risk of progressing to end-stage renal disease. Because of this risk, new therapeutic strategies are needed for steroid-resistant nephrotic syndrome. These strategies have historically focused on identifying effective alternative immunosuppressive agents, such as ciclosporin and tacrolimus, yet evidence now indicates that nephrotic syndrome results from podocyte dysfunction. Even conventional immunosuppressive agents, such as glucocorticoids and ciclosporin, directly affect podocyte structure and function, challenging the 'immune theory' of the pathogenesis of childhood nephrotic syndrome in which disease is caused by T cells. This Review summarizes the currently available treatments for childhood nephrotic syndrome, and discusses selected novel pathways in podocytes that could be targeted for the development of next-generation treatments for children with this syndrome.

Comparison of direct action of thiazolidinediones and glucocorticoids on renal podocytes: protection from injury and molecular effects.

The U.S. Food and Drug Administration-approved thiazolidinediones pioglitazone and rosiglitazone are peroxisome proliferator-activated receptor-γ (PPARγ) agonists developed to control serum glucose in patients with diabetes. They have been found to reduce proteinuria and microalbuminuria in both diabetic nephropathy and nondiabetic glomerulosclerosis. We hypothesized that the renal protective effects of thiazolidinediones result, at least in part, from their direct action on podocytes, similar to glucocorticoids. Treatment with pioglitazone, rosiglitazone, or dexamethasone significantly protected podocytes against puromycin aminonucleoside-induced injury (designed to mimic nephrotic syndrome-related injury), as determined by both cell survival and actin cytoskeletal integrity. Furthermore, we compared the ability of these drugs to modulate key signaling pathways in podocytes that may be critical to their protective effects. Rosiglitazone deactivated the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1/2, p38 MAPK, and stress-activated protein kinase/c-Jun NH2-terminal kinase, whereas pioglitazone did not, and dexamethasone deactivated to some extent. Similar to dexamethasone, both thiazolidinediones increased the glucocorticoid receptor phosphorylation, and this response to rosiglitazone and possibly to pioglitazone was PPARγ-dependent. Furthermore, both drugs mimicked or enhanced the effects of dexamethasone on glucocorticoid-responsive genes in a PPARγ- and glucocorticoid receptor-dependent manner. In addition, both thiazolidinediones mimicked dexamethasone-induced effects on calcineurin activity. In summary, thiazolidinediones are able to modulate the glucocorticoid pathway and exert direct protective effects on podocytes, similar to glucocorticoids. This suggests that thiazolidinediones may have potential clinical utility as either primary or adjunctive therapy for nephrotic syndrome or other diseases treated with glucocorticoids. These findings may also lend mechanistic insight into the well established but poorly understood renal protective effects of thiazolidinediones in diabetic nephropathy.

Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury.

While mitogen-activated protein kinase (MAPK) activation has been implicated in the pathogenesis of various glomerular diseases, including nephrotic syndrome (NS), its specific role in podocyte injury is not known. We hypothesized that MK-2, a downstream substrate of p38 MAPK, mediates the adverse effects of this pathway and that inhibition of MK-2 would protect podocytes from NS-related injury. Using cultured podocytes, we analyzed 1) the roles of MK-2 and p38 MAPK in puromycin aminonucleoside (PAN)-induced podocyte injury; 2) the ability of specific MK-2 and p38 MAPK inhibitors to protect podocytes against injury; 3) the role of serum albumin, known to induce podocyte injury, in activating p38 MAPK/MK-2 signaling; and 4) the role of p38 MAPK/MK-2 signaling in the expression of Cox-2, an enzyme associated with podocyte injury. Treatment with protein kinase inhibitors specific for both MK-2 (C23, a pyrrolopyridine-type compound) or p38 MAPK (SB203580) reduced PAN-induced podocyte injury and actin cytoskeletal disruption. Both inhibitors reduced baseline podocyte p38 MAPK/MK-2 signaling, as measured by the degree of phosphorylation of HSPB1, a downstream substrate of MK-2, but exhibited disparate effects on upstream signaling. Serum albumin activated p38 MAPK/MK-2 signaling and induced Cox-2 expression, and these responses were blocked by both inhibitors. Given the critical importance of podocyte injury to both NS and other progressive glomerular diseases, these data suggest an important role for p38 MAPK/MK-2 signaling in podocyte injury and identify MK-2 inhibition as a promising potential therapeutic strategy to protect podocytes in various glomerular diseases.

Dose- and time-dependent glucocorticoid receptor signaling in podocytes.

Glucocorticoids (GC) are the primary therapy for idiopathic nephrotic syndrome (NS). Recent evidence has identified glomerular podocytes as a potential site of GC action in this disease. The objectives of this study were to determine the presence of key components of the glucocorticoid receptor (GR) complex and the functionality of this signaling pathway in podocytes and to explore potential opportunities for manipulation of GC responsiveness. Here, we show that cultured murine podocytes express key components of the GR complex, including the GR, heat shock protein 90, and the immunophilins FKBP51 and FKBP52. The functionality of GR-mediated signaling was verified by measuring several GC (dexamethasone)-induced responses, including 1) increases in mRNA and protein levels of selected GC-regulated genes (FKBP51, phenol sulfotransferase 1, αB-crystallin); 2) downregulation of the GR protein; 3) increased phosphorylation of the GR; and 4) translocation of the GR into the nuclear fraction. Dexamethasone-induced phosphorylation and downregulation of GR protein were also demonstrated in isolated rat glomeruli. Podocyte gene expression in response to dexamethasone was regulated at both the transcriptional and posttranscriptional levels, the latter also including protein degradation. Short-term, high-dose GC treatment resulted in similar changes in gene expression and GR phosphorylation to that of long-term, low-dose GC treatment, thus providing a molecular rationale for the known efficacy of pulse GC therapy in NS. Induction of FKBP51 and downregulation of the GR represent negative feedback mechanisms that can potentially be exploited to improve clinical GC efficacy. Collectively, these findings demonstrate the presence of key molecular components of the GR signaling pathway and its functionality in podocytes and identify novel opportunities for improving clinical GC efficacy in the treatment of NS.

Abnormal interaction of motor neuropathy-associated mutant HspB8 (Hsp22) forms with the RNA helicase Ddx20 (gemin3).

A number of missense mutations in the two related small heat shock proteins HspB8 (Hsp22) and HspB1 (Hsp27) have been associated with the inherited motor neuron diseases (MND) distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 and HspB1 interact with each other, suggesting that these two etiologic factors may act through a common biochemical mechanism. However, their role in neuron biology and in MND is not understood. In a yeast two-hybrid screen, we identified the DEAD box protein Ddx20 (gemin3, DP103) as interacting partner of HspB8. Using co-immunoprecipitation, chemical cross-linking, and in vivo quantitative fluorescence resonance energy transfer, we confirmed this interaction. We also show that the two disease-associated mutant HspB8 forms have abnormally increased binding to Ddx20. Ddx20 itself binds to the survival-of-motor-neurons protein (SMN protein), and mutations in the SMN1 gene cause spinal muscular atrophy, another MND and one of the most prevalent genetic causes of infant mortality. Thus, these protein interaction data have linked the three etiologic factors HspB8, HspB1, and SMN protein, and mutations in any of their genes cause the various forms of MND. Ddx20 and SMN protein are involved in spliceosome assembly and pre-mRNA processing. RNase treatment affected the interaction of the mutant HspB8 with Ddx20 suggesting RNA involvement in this interaction and a potential role of HspB8 in ribonucleoprotein processing.

Myopathy-associated alphaB-crystallin mutants: abnormal phosphorylation, intracellular location, and interactions with other small heat shock proteins.

Three mutations (R120G, Q151X, and 464delCT) in the small heat shock protein alphaB-crystallin cause inherited myofibrillar myopathy. In an effort to elucidate the molecular basis for the associated myopathy, we have determined the following for these mutant alphaB-crystallin proteins: (i) the formation of aggregates in transfected cells; (ii) the partition into different subcellular fractions; (iii) the phosphorylation status; and (iv) the ability to interact with themselves, with wild-typealphaB-crystallin, and with other small heat shock proteins that are abundant in muscles. We found that all three alphaB-crystallin mutants have an increased tendency to form cytoplasmic aggregates in transfected cells and significantly increased levels of phosphorylation when compared with the wild-type protein. Although wild-type alphaB-crystallin partitioned essentially into the cytosol and membranes/organelles fractions, mutant alphaB-crystallin proteins partitioned additionally into the nuclear and cytoskeletal fractions. By using various protein interaction assays, including quantitative fluorescence resonance energy transfer measurements in live cells, we found abnormal interactions of the various alphaB-crystallin mutants with wild-type alphaB-crystallin, with themselves, and with the other small heat shock proteins Hsp20, Hsp22, and possibly with Hsp27. The collected data suggest that eachalphaB-crystallin mutant has a unique pattern of abnormal interaction properties. These distinct properties of the alphaB-crystallin mutants identified are likely to contribute to a better understanding of the gradual manifestation and clinical heterogeneity of the associated myopathy in patients.

Induction of Hsp22 (HspB8) by estrogen and the metalloestrogen cadmium in estrogen receptor-positive breast cancer cells.

Estrogen (E2) plays a critical role in the etiology and progression of human breast cancer. The estrogenic response is complex and not completely understood, including in terms of the involved responsive genes. Here we show that Hsp22 (synonyms: HspB8, E2lG1, H11), a member of the small heat shock protein (sHSP) superfamily, was induced by E2 in estrogen receptor-positive MCF-7 breast cancer cells, resulting in an elevated Hsp22 protein level, whereas it was not induced in estrogen receptor-negative MDA-MB-231 cells. This induction was prevented by the pure anti-estrogen ICI182780 (faslodex, fulvestrant), whereas tamoxifen, a substance with mixed estrogenic and antiestrogenic properties, had no major inhibitory effect on this induction, nor did it induce Hsp22 on its own. Cadmium (Cd) is an environmental pollutant with estrogenic properties (metalloestrogen) that has been implicated in breast cancer. Treatment of MCF-7 cells with Cd also resulted in induction of Hsp22, and this induction was also inhibited by ICI182780. In live MCF-7 cells, Hsp22 interacted at the level of dimers with Hsp27, a related sHSP, as was shown by quantitative fluorescence resonance energy transfer measurements. In cytosolic extracts of MCF-7 cells, most of the E2- and Cd-induced Hsp22 was incorporated into high-molecular mass complexes. In part, Hsp22 and Hsp27 were components of distinct populations of these complexes. Finally, candidate elements in the Hsp22 promoter were identified by sequence analysis that could account for the induction of Hsp22 by E2 and Cd. Taken together, Hsp22 induction represents a new aspect of the estrogenic response with potential significance for the biology of estrogen receptor-positive breast cancer cells.

Abnormal small heat shock protein interactions involving neuropathy-associated HSP22 (HSPB8) mutants.

Two mutations (K141E, K141N) in the small heat shock protein (sHSP) HSP22 (HSPB8) are associated with the inherited peripheral motor neuron disorders distal hereditary motor neuropathy type II and axonal Charcot-Marie-Tooth disease type 2L. HSP22 is known to form homodimers, heterodimers with other sHSPs, and larger oligomers. In an effort to elucidate the cellular basis for these diseases, we have determined the ability of mutant HSP22 to interact with itself, with wild-type HSP22, and with other sHSPs that are abundant in neurons. Using the yeast two-hybrid method, quantitative fluorescence resonance energy transfer in live cells, and cross-linking, we found aberrantly increased interactions of mutant HSP22 forms with themselves, with wild-type HSP22, and with the other sHSPs, alphaB-crystallin, and HSP27. Interaction with HSP20 was not affected by the mutations. The data suggest that each mutant form of HSP22 has a characteristic pattern of abnormal interaction properties. A mutation (S135F) in HSP27 that is also associated with these disorders showed increased interaction with wild-type HSP22 also, suggesting linkage of these two etiologic factors, HSP22 and HSP27, into one common pathway. Increased interactions involving mutant sHSPs may be the molecular basis for their increased tendency to form cytoplasmic protein aggregates, and for the occurrence of the associated neuropathies.

Conformational changes resulting from pseudophosphorylation of mammalian small heat shock proteins--a two-hybrid study.

The human genome codes for 10 so-called mammalian small heat shock or stress proteins (sHsp) with the various tissues expressing characteristic sets of sHsps. Most sHsps interact with each other and form homo- and heterooligomeric complexes. Some of the sHsps are phosphoproteins in vivo, and phosphorylation has been implicated in the regulation of complex size and composition. In this study, we analyze, by the 2-hybrid method, the reporter gene activation pattern of several sHsp pairs that previously have been demonstrated to interact. We show that pseudophosphorylation (mimicry of phosphorylation) of the homologous phosphorylation sites Ser15 and Ser16 in Hsp27 and Hsp20, respectively, modulates characteristics of these sHsps that can be detected by their ability to activate reporter genes in suitable 2-hybrid assays. Pseudophosphorylation of the separated N-terminus of Hsp27 alone is not sufficient for the activation of the reporter genes, whereas the separated C-terminus is sufficient. We conclude that pseudophosphorylation of Hsp27 and Hsp20 at their N-termini results in conformational changes that can be detected by their interaction with other sHsps. Pseudophosphorylation of alphaB-crystallin at Ser19, in contrast, had no detectable consequences.

Interactions of HSP22 (HSPB8) with HSP20, alphaB-crystallin, and HSPB3.

Seven of the 10 mammalian small heat shock proteins (sHSP) are expressed in muscle where they constitute 3% or more of total protein. sHSPs interact with one another, and these interactions are believed to be important for their functions. In cell types expressing multiple sHSPs, it is of interest to know which sHSPs interact with one another. We have previously shown that HSP22 interacts with itself as well as with HSP27, MKBP, and cvHSP. Using yeast two-hybrid assays and Förster resonance energy transfer microscopy, we now show that HSP22 also can interact with two additional members of the sHSP family, alphaB-crystallin and HSP20. We also show that HSP22 is found in HPLC fractions of primate cardiac muscle containing high molecular weight complexes that include alphaB-crystallin and HSP20. Our results suggest that a variety of oligomers composed of different proportions of different sHSPs may form in cell types expressing multiple sHSPs.

p38 MAPK/HSP25 signaling mediates cadmium-induced contraction of mesangial cells and renal glomeruli.

The environmental pollutant cadmium affects human health, with the kidney being a primary target. In addition to proximal tubules, glomeruli and their contractile mesangial cells have also been identified as targets of cadmium nephrotoxicity. Glomerular contraction is thought to contribute to reduced glomerular filtration, a characteristic of cadmium nephrotoxicity. Because p38 MAPK/HSP25 signaling has been implicated in smooth muscle contraction, we examined its role in cadmium-induced contraction of mesangial cells. We report that exposure of mesangial cells to cadmium resulted in 1) cell contraction, 2) activation of MAP kinases, 3) increased HSP25 phosphorylation coincident with p38 MAP kinase activation, 4) sequential phosphorylation of the two phosphorylation sites of mouse HSP25 with Ser15 being phosphorylated before Ser86, 5) reduction of oligomeric size of HSP25, and 6) association of HSP25 with microfilaments. Exposure of isolated rat glomeruli to cadmium also resulted in contraction and increased HSP25 phosphorylation. The cadmium-induced responses were inhibited by the specific p38 MAP kinase inhibitor SB-203580, and cadmium-induced phosphorylation of HSP25 was inhibited by expression of a dominant-negative p38 MAP kinase mutant. These findings tentatively suggest that cadmium-induced nephrotoxicity results, in part, from glomerular contraction due to p38 MAP kinase/HSP25 signaling-dependent contraction of mesangial cells. With regard to the cellular action of HSP25, these data support a change in paradigm: in addition to its well-established cytoprotective function, HSP25 may also be involved in processes that ultimately lead to adverse effects, as is observed in the response of mesangial cells to cadmium.

Interaction of human HSP22 (HSPB8) with other small heat shock proteins.

Mammalian small heat shock proteins (sHSP) are abundant in muscles and are implicated in both muscle function and myopathies. Recently a new sHSP, HSP22 (HSPB8, H11), was identified in the human heart by its interaction with HSP27 (HSPB1). Using phylogenetic analysis we show that HSP22 is a true member of the sHSP superfamily. sHSPs interact with each other and form homo- and hetero-oligomeric complexes. The function of these complexes is poorly understood. Using gel filtration HPLC, the yeast two-hybrid method, immunoprecipitation, cross-linking, and fluorescence resonance energy transfer microscopy, we report that (i). HSP22 forms high molecular mass complexes in the heart, (ii). HSP22 interacts with itself, cvHSP (HSPB7), MKBP (HSPB2) and HSP27, and (iii). HSP22 has two binding domains (N- and C-terminal) that are specific for different binding partners. HSP22 homo-dimers are formed through N-N and N-C interactions, and HSP22-cvHSP hetero-dimers through C-C interaction. HSP22-MKBP and HSP22-HSP27 hetero-dimers involve the N and C termini of HSP22 and HSP27, respectively, but appear to require full-length protein as a binding partner.

The sperm outer dense fiber protein is the 10th member of the superfamily of mammalian small stress proteins.

Nine proteins have been assigned to date to the superfamily of mammalian small heat shock proteins (sHsps): Hsp27 (HspB1, Hsp25), myotonic dystrophy protein kinase-binding protein (MKBP) (HspB2), HspB3, alphaA-crystallin (HspB4), alphaB-crystallin (HspB5), Hsp20 (p20, HspB6), cardiovascular heat shock protein (cvHsp [HspB7]), Hsp22 (HspB8), and HspB9. The most pronounced structural feature of sHsps is the alpha-crystallin domain, a conserved stretch of approximately 80 amino acid residues in the C-terminal half of the molecule. Using the alpha-crystallin domain of human Hsp27 as query in a BLAST search, we found sequence similarity with another mammalian protein, the sperm outer dense fiber protein (ODFP). ODFP occurs exclusively in the axoneme of sperm cells. Multiple alignment of human ODFP with the other human sHsps reveals that the primary structure of ODFP fits into the sequence pattern that is typical for this protein superfamily: alpha-crystallin domain (conserved), N-terminal domain (less conserved), central region (variable), and C-terminal tails (variable). In a phylogenetic analysis of 167 proteins of the sHsp superfamily, using Bayesian inference, mammalian ODFPs form a clade and are nested within previously identified sHsps, some of which have been implicated in cytoskeletal functions. Both the multiple alignment and the phylogeny suggest that ODFP is the 10th member of the superfamily of mammalian sHsps, and we propose to name it HspB10 in analogy with the other sHsps. The C-terminal tail of HspB10 has a remarkable low-complexity structure consisting of 10 repeats of the motif C-X-P. A BLAST search using the C-terminal tail as query revealed similarity with sequence elements in a number of Drosophila male sperm proteins, and mammalian type I keratins and cornifin-alpha. Taken together, the following findings suggest a specialized role of HspB10 in cytoskeleton: (1) the exclusive location in sperm cell tails, (2) the phylogenetic relationship with sHsps implicated in cytoskeletal functions, and (3) the partial similarity with cytoskeletal proteins.