Pivotal role of the C2 domain of the Smurf1 ubiquitin ligase in substrate selection

The C2-WW-HECT-type ubiquitin ligases Smurf1 and Smurf2 play a critical role in embryogenesis and adult bone homeostasis via regulation of bone morphogenetic protein, Wnt, and RhoA signaling pathways. The intramolecular interaction between C2 and HECT domains autoinhibits the ligase activity of Smurf2. However, the role of the Smurf1 C2 domain remains elusive. Here, we show that the C2-HECT autoinhibition mechanism is not observed in Smurf1, and instead its C2 domain functions in substrate selection. The Smurf1 C2 domain exerts a key role in localization to the plasma membrane and endows Smurf1 with differential activity toward RhoA versus Smad5 and Runx2. Crystal structure analysis reveals that the Smurf1 C2 domain possesses a typical anti-parallel β-sandwich fold. Examination of the sulfate-binding site analysis reveals two key lysine residues, Lys-28 and Lys-85, within the C2 domain that are important for Smurf1 localization at the plasma membrane, regulation on cell migration, and robust ligase activity toward RhoA, which further supports a Ca(2+)-independent localization mechanism for Smurf1. These findings demonstrate a previously unidentified role of the Smurf1 C2 domain in substrate selection and cellular localization.     

Distinct roles of FANCO/RAD51C protein in DNA damage signaling and repair: implications for Fanconi anemia and breast cancer susceptibility

RAD51C, a RAD51 paralog, has been implicated in homologous recombination (HR), and germ line mutations in RAD51C are known to cause Fanconi anemia (FA)-like disorder and breast and ovarian cancers. The role of RAD51C in the FA pathway of DNA interstrand cross-link (ICL) repair and as a tumor suppressor is obscure. Here, we report that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G(2)/M accumulation, which are hallmarks of the FA phenotype. We find that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrate that RAD51C plays a vital role in the HR-mediated repair of DNA lesions associated with replication. Finally, we show that RAD51C participates in ICL and double strand break-induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C that were identified in FA and breast and ovarian cancers reveal that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor.     

Protein kinase C: Mediator or inhibitor of insulin action?

The role of protein kinase C in insulin signal transduction i s controversial It has been postulated that protein kinase C i s activated by insulin and that the kinase i s directly involved in insulin-mediated metabolic processes. In opposition to this view i s the hypothesis that protein kinase C is not activated by insulin and, more importantly, may be responsible for attenuation of the insulin signal The evidence for and against protein kinase C as a mediator of the insulin signal will be put in perspective followed by discussion of the possible role of the kinase in the pathogenesis of insulin resistance in type II diabetes.     

Membrane attack complex of complement and neutrophils mediate the injury of acid aspiration.

A significant role for the alternative complement pathway in acid aspiration has been demonstrated by the observation that C3 genetic knockout mice are protected from injury. Utilizing C5-deficient mice, we now test the role of the terminal complement components in mediating injury. Lung permeability in C5-deficient mice was 64% less than in wild-type animals and was similar to wild-type mice treated with soluble complement receptor type 1, which gave a 67% protection. Injury was fully restored in C5-deficient mice reconstituted with wild-type serum. The role of neutrophils was established in immunodepleted wild-type animals that showed a 58% protection. Injury was further reduced (90%) with the addition of soluble complement receptor type 1, indicating an additive effect of neutrophils and complement. Similarly, an additional protection was noted in C5-deficient neutropenic mice, indicating that neutrophil-mediated injury does not require C5a. Thus acid aspiration injury is mediated by the membrane attack complex and neutrophils. Neutrophil activity is independent of C5a.     

Hydrophilic domains of scaffolding protein CbpA promote glycosyl hydrolase activity and localization of cellulosomes to the cell surface of Clostridium cellulovorans

CbpA, the scaffolding protein of Clostridium cellulovorans cellulosomes, possesses one family 3 cellulose binding domain, nine cohesin domains, and four hydrophilic domains (HLDs). Among the three types of domains, the function of the HLDs is still unknown. We proposed previously that the HLDs of CbpA play a role in attaching the cellulosome to the cell surface, since they showed some homology to the surface layer homology domains of EngE. Several recombinant proteins with HLDs (rHLDs) and recombinant EngE (rEngE) were examined to determine their binding to the C. cellulovorans cell wall fraction. Tandemly linked rHLDs showed higher affinity for the cell wall than individual rHLDs showed. EngE was shown to have a higher affinity for cell walls than rHLDs have. C. cellulovorans native cellulosomes were found to have higher affinity for cell walls than rHLDs have. When immunoblot analysis was carried out with the native cellulosome fraction bound to cell wall fragments, the presence of EngE was also confirmed, suggesting that the mechanism anchoring CbpA to the C. cellulovorans cell surface was mediated through EngE and that the HLDs play a secondary role in the attachment of the cellulosome to the cell surface. During a study of the role of HLDs on cellulose degradation, the mini-cellulosome complexes with HLDs degraded cellulose more efficiently than complexes without HLDs degraded cellulose. The rHLDs also showed binding affinity for crystalline cellulose and carboxymethyl cellulose. These results suggest that the CbpA HLDs play a major role and a minor role in C. cellulovorans cellulosomes. The primary role increases cellulose degradation activity by binding the cellulosome complex to the cellulose substrate; secondarily, HLDs aid the binding of the CbpA/cellulosome to the C. cellulovorans cell surface.     

Control of voluntary movements mediated via propriospinal neurons

Cortico- and rubrospinal tracts play an important role in controlling voluntary movements. Transection of these tracts in different spinal cord layers gives different effects that may be explained by the influence of different spinal cord neuronal networks. The aim of the present work was to study the role of C3/C4 propriospinal system in movement control and processes of motor recovery. It was shown that propriospinal system C3/C4 play crucial role in motor recovery after lesion of cortico- and rubrospinal tracts in C5, whereas ventrally located tracts are important after the same lesion in C2. More over, propriospinal system C3/C4 can mediate the command for some voluntary movements in cats.     

Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C

Ubiquitin-mediated proteolysis is critical for the alternation between DNA replication and mitosis and for the key regulatory events in mitosis. The anaphase-promoting complex/cyclosome (APC/C) is a conserved ubiquitin ligase that has a fundamental role in regulating mitosis and the cell cycle in all eukaryotes. In vertebrate cells, early mitotic inhibitor 1 (Emi1) has been proposed as an important APC/C inhibitor whose destruction may trigger activation of the APC/C at mitosis. However, in this study, we show that the degradation of Emi1 is not required to activate the APC/C in mitosis. Instead, we uncover a key role for Emi1 in inhibiting the APC/C in interphase to stabilize the mitotic cyclins and geminin to promote mitosis and prevent rereplication. Thus, Emi1 plays a crucial role in the cell cycle to couple DNA replication with mitosis, and our results also question the current view that the APC/C has to be inactivated to allow DNA replication.     

Opposing regulatory roles of complement factor 5 in the development of bleomycin-induced pulmonary fibrosis

The mechanisms of idiopathic pulmonary fibrosis pathogenesis, a chronic and progressive interstitial lung disease, remain elusive. The complement system, a crucial arm of the innate immune response, plays a pivotal role in several pathological disorders; however, the contribution of individual complement components to lung fibrosis has not yet been examined. Complement factor 5 (C5) and its cleavage product C5a are critical mediators in inflammatory diseases. Thus, to evaluate the role of C5 in lung fibrosis, we compared congenic C5-sufficient and C5-deficient mice in a well-characterized murine model of bleomycin-induced pulmonary fibrosis. C5-deficient mice had an exaggerated inflammatory phenotype compared with C5-sufficient mice during acute bleomycin-induced lung injury. These findings suggest a protective and anti-inflammatory role for C5, which was linked to the regulation of matrix metalloproteinases involved in cell migration. In contrast, C5 had a detrimental effect during chronic stages of bleomycin-induced injury, indicating a profibrotic role for C5. This deleterious activity for C5 was associated with expression of the fibrogenic cytokine TGF-beta1 and matrix metalloproteinase-3, an important mediator in fibroblast contraction. Altogether, our data reveal novel and opposing roles for C5 in both inflammation and tissue repair. Furthermore, these findings provide insight into the development of new therapeutic strategies for idiopathic pulmonary fibrosis patients.     

The Role of the Anaphase-Promoting Complex/Cyclosome in Plant Growth

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that plays a major role in the progression of the eukaryotic cell cycle. This unusual protein complex targets key cell cycle regulators, such as mitotic cyclins and securins, for degradation via the 26S proteasome by ubiquitination, triggering the metaphase-to-anaphase transition and exit from mitosis. Because of its essential role in cell cycle regulation, the APC/C has been extensively studied in mammals and yeasts, but relatively less in plants. Evidence shows that, besides its well-known role in cell cycle regulation, the APC/C also has functions beyond the cell cycle. In metazoans, the APC/C has been implicated in cell differentiation, disease control, basic metabolism and neuronal survival. Recent studies also have shed light on specific functions of the APC/C during plant development. Plant APC/C subunits and activators have been reported to play a role in cellular differentiation, vascular development, shoot branching, female and male gametophyte development and embryogenesis. Here, we discuss our current understanding of the APC/C controlling plant growth.     

Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.     

Crystal structure of the restriction-modification system control element C.Bcll and mapping of its binding site

Protection from DNA invasion is afforded by restriction-modification systems in many bacteria. The efficiency of protection depends crucially on the relative expression levels of restriction versus methytransferase genes. This regulation is provided by a controller protein, named C protein. Studies of the Bcll system in E. coli suggest that C.Bcll functions as a negative regulator for M.Bcll expression, implying that it plays a role in defense against foreign DNA during virus infection. C.Bcll binds (Kd = 14.3 nM) to a 2-fold symmetric C box DNA sequence that overlaps with the putative -35 promoter region upstream of the bcllM and bcllC genes. The C.Bcll fold comprises five alpha helices: two helices form a helix-turn-helix motif, and the remaining three helices form the extensive dimer interface. The C.Bcll-DNA model proposed suggests that DNA bending might play an important role in gene regulation, and that Glu27 and Asp31 in C.Bcll might function critically in the regulation.     

Cytochrome p450 2C (CYP2C) in ischemic heart injury and vascular dysfunction

The cytochrome p450 2C (CYP2C) monooxygenase family is a key player in the generation of epoxyeicosatrienoic acids. It has recently become apparent that CYP plays an important role in cardiovascular physiology and contributes to the pathogenesis of various cardiovascular diseases. In particular, several studies have demonstrated a role for these enzymes in cardiac ischemia and reperfusion injury. The current review summarizes the role of the CYP epoxygenase, CYP2C9, in ischemic heart disease and vascular homeostasis.     

Critical role of the cysteine 323 residue in the catalytic activity of human glutamate dehydrogenase isozymes

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the Km of the enzyme for NADH and alpha-ketoglutarate increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency (kcat/Km) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in kcat values. There was a linear relationship between incorporation of [14C]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [14C] incorporated per mol of monomer for wild type hGDHs. No incorporation of [14C]p-chloromer-curibenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.     

Modulation of the immune response by anaphylatoxin in the microenvironment of the interacting cells

It was shown that the anaphylatoxins C3a and C5a can modulate in vitro immunological reactivities. C3a suppresses both the in vitro polyclonal antibody response and the specific antibody response to sheep red blood cells (SRBC) of both mouse spleen cells and human peripheral blood cells. The target cell in the mouse for C3a appears to be an Lyt-1+2- suppressor-inducer cell and macrophages appear not to be required. In contrast to C3a, C5a enhances in vitro responses of mice. Both the response to SRBC and the mixed lymphocyte reaction are enhanced by C5a. This enhancement appears to be through an Ia- macrophage that contains receptors for C5a. It appears that enhancement may be brought about by interleukin 1, which is released when Ia- macrophages are pulsed with C5a. It is suggested that these anaphylatoxins, when present in high concentrations in the microenvironment of the interacting cells of the immune system, play a dynamic role in the regulation of the immune response. Peptide fragments cleaved from the Fc portion of antibody, complexed with antigen in this microenvironment, may have a similar regulating role.     

Insights into folate/FAD-dependent tRNA methyltransferase mechanism: role of two highly conserved cysteines in catalysis

The flavoprotein TrmFO methylates specifically the C5 carbon of the highly conserved uridine 54 in tRNAs. Contrary to most methyltransferases, the 1-carbon unit transferred by TrmFO derives from 5,10-methylenetetrahydrofolate and not from S-adenosyl-L-methionine. The enzyme also employs the FAD hydroquinone as a reducing agent of the C5 methylene U54-tRNA intermediate in vitro. By analogy with the catalytic mechanism of thymidylate synthase ThyA, a conserved cysteine located near the FAD isoalloxazine ring was proposed to act as a nucleophile during catalysis. Here, we mutated this residue (Cys-53 in Bacillus subtilis TrmFO) to alanine and investigated its functional role. Biophysical characterization of this variant demonstrated the major structural role of Cys-53 in maintaining both the integrity and plasticity of the flavin binding site. Unexpectedly, gel mobility shift assays showed that, like the wild-type enzyme, the inactive C53A variant was capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. This result confirms the existence of a covalent intermediate during catalysis but rules out a nucleophilic role for Cys-53. To identify the actual nucleophile, two other strictly conserved cysteines (Cys-192 and Cys-226) that are relatively far from the active site were replaced with alanine, and a double mutant C53A/C226A was generated. Interestingly, only mutations that target Cys-226 impeded TrmFO from forming a covalent complex and methylating tRNA. Altogether, we propose a revised mechanism for the m(5)U54 modification catalyzed by TrmFO, where Cys-226 attacks the C6 atom of the uridine, and Cys-53 plays the role of the general base abstracting the C5 proton.     

Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.     

Requirement of heat-labile opsonins for maximal phagocytosis of Candida albicans.

The requirement for heat-labile opsonins for phagocytosis of 2 serologically distinct strains and a number of clinical isolates of Candida albicans was examined by a previously described radiometric technique. The results indicated that both strains and all 10 isolates of C. albicans examined required heat-labile opsonins for maximal phagocytosis. The data suggest that heat-labile opsonins play an important role in immunity to C. albicans. However, the possibility that some strains of C. albicans may not utilize heat-labile opsonins cannot be excluded.