Rainer Benndorf

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

Abstract 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, αA-crystallin (HspB4), αB-crystallin (HspB5), Hsp20 (p20, HspB6), cardiovascular heat shock protein (cvHsp [HspB7]), Hsp22 (HspB8), and HspB9. The most pronounced structural feature of sHsps is the α-crystallin domain, a conserved stretch of approximately 80 amino acid residues in the C-terminal half of the molecule. Using the α-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: α-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-α. 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.

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.

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, αB‐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.—Jean‐Marc Fontaine, Xiankui Sun, Adam D. Hoppe, Stephanie Simon, Patrick Vicart, Michael J. Welsh, and Rainer Benndorf. Abnormal small heat shock protein interactions involving neuropathy‐associated HSP22 (HSPB8) mutants. FASEB J. 20,E1579–E1588 (2006)

HSP27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCK-A cells

We investigated how heat shock protein 27 (HSP27) and its phosphorylation are involved in the action of cholecystokinin (CCK) on the actin cytoskeleton by genetic manipulation of Chinese hamster ovary (CHO) cells stably transfected with the CCK-A receptor. In these cells, as in rat acini, CCK activated p38 mitogen-activated protein (MAP) kinase and increased the phosphorylation of HSP27. This effect could be blocked with the p38 MAP kinase inhibitor SB-203580. Examination by confocal microscopy of cells stained with rhodamine phalloidin showed that CCK dose-dependently induced changes of the actin cytoskeleton, including cell shape changes, which were coincident with actin cytoskeleton fragmentation and formation of actin filament patches in the cells. To further evaluate the role of HSP27, CHO-CCK-A cells were transfected with expression vectors for either wild-type (wt) or mutant (3A, 3G, and 3D) human HSP27. Overexpression of wt-HSP27 and 3D-HSP27 inhibited the effects on the actin cytoskeleton seen after high-dose CCK stimulation. In contrast, overexpression of nonphosphorylatable mutants, 3A- and 3G-HSP27, or inhibition of phosphorylation of HSP27 by preincubation of wt-HSP27 transfected cells with SB-203580 did not protect the actin cytoskeleton. These results suggest that phosphorylation of HSP27 is required to stabilize the actin cytoskeleton and to protect the cells from the effects of high concentrations of CCK.

The effect of the intersubunit disulfide bond on the structural and functional properties of the small heat shock protein Hsp25

The murine small heat shock protein Hsp25 carries a single cysteine residue in position 141 of its amino acid sequence. Interestingly, Hsp25 can exist within the cell as covalently bound dimer which is linked by an intermolecular disulfide bond between two monomers. Oxidative stress caused by treatment of the cells with diamide, arsenite, or hydrogen peroxide leads to an increase in Hsp25-dimerisation which can be blocked by simultaneous treatment with reducing agents. Recombinant Hsp25 was prepared in an oxidized dimeric (oxHsp25) and reduced monomeric (redHsp25) from. The two species were compared with regard to secondary structure, stability, oligomerization properties and their chaperone activity. It is demonstrated by CD measurements in the far UV region that there are no significant differences in the secondary structure and temperature- or pH-stability of oxHsp25 and redHsp25. However, according to CD measurements in the near UV region an increase in the asymmetry of the microenvironment of aromatic residues in oxHsp25 is observed. Furthermore, an increase in stability of the hydrophobic environment of the tryptophan residues mainly located in the N-terminal domain of the protein against urea denaturation is detected in oxHsp25. Both reduced and oxidized Hsp25 from oligomeric complexes of similar size and stability against detergents and both species prevent thermal aggregation of citrate synthase and assist significantly in oxaloacetic acid-induced refolding of the enzyme. Hence, the overall secondary structure, the degree of oligomerization and the chaperone activity of Hsp25 seem independent of the formation of the intermolecular disulfide bond and only the stability of the hydrophobic N-terminal part of the molecule is influenced by formation of this bound. The obtained data do not exclude the possible involvement of dimerization of this protein in other cellular functions, e.g. in intracellular sulfhydryl-buffering or in the protection of actin filaments from fragmentation upon oxidative stress.

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.

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.

Mammalian protein homologous to VAT‐1 of Torpedo californica: Isolation from Ehrlich ascites tumor cells, biochemical characterization, and organization of its gene

Recently, interest has focused on the human gene encoding the putative protein homologous to VAT‐1, the major protein of the synaptic vesicles of the electric organ of the Pacific electric ray Torpedo californica, after it has been localized on chromosome locus 17q21 in a region encompassing the breast cancer gene BRCA1. Chromosomal instability in this region is implicated in inherited predisposition for breast and ovarian cancer. Here we describe isolation and biochemical characterization of a mammalian 48 kDa protein homologous to the VAT‐1 protein of Torpedo californica. This VAT‐1 homolog was isolated from a murine breast cancer cell line (Ehrlich ascites tumor) and identified by sequencing of cleavage peptides. The isolated VAT‐1 homolog protein displays an ATPase activity and exists in two isoforms with isoelectric points of 5.7 and 5.8. cDNA was prepared from Ehrlich ascites tumor cells, and the murine VAT‐1 homolog sequence was amplified by polymerase chain reaction and partially sequenced. The known part of the murine and the human translated sequences share 97% identity. By Northern blots, the size of the VAT‐1 homolog mRNA in both murine and human (T47D) breast cancer cells was determined to be 2.8 kb. Based on the presented data, a modified gene structure of the human VAT‐1 homolog with an extended exon 1 is proposed. VAT‐1 and the mammalian VAT‐1 homolog form a subgroup within the protein superfamily of medium‐chain dehydrogenases/reductases. J. Cell. Biochem. 69:304–315, 1998.

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.