Autolysis of a novel multidomain subtilase-cold-adapted deseasin MCP-01 is pH-dependent and the surface loops in its catalytic domain, the linker, and the P_proprotein domain are susceptible to proteolytic attack

Cold-adapted deseasin MCP-01 is a novel type subtilase with a multidomain structure containing a catalytic domain, a linker, a P_proprotein domain, and a PKD domain. Its autolysis was pH-dependent due to its flexible structure. N-terminal sequence analysis of the autolytic peptides revealed four autolytic sites in the catalytic domain. Three of these are in the same loops as mesophilic subtilases and one is unlike anything previously reported. Two autolytic sites were deduced in its linker and three in its P_proprotein domain, indicating the linker and the P_proprotein domain are flexible and susceptible to proteolytic attacks. Therefore, during MCP-01 autolysis, the linker and the P_proprotein domain of MCP-01 were easily attacked by proteolysis, resulting in cleavage of the C-terminal region. At the same time, some autolytic sites in the surface loops of the catalytic domain were cleaved. This is the first report describing the autolytic mechanism of a multidomain subtilase.     

The contribution of proline residues to protein stability is associated with isomerization equilibrium in both unfolded and folded states

It has long been understood that the proline residue has lower configurational entropy than any other amino acid residue due to pyrrolidine ring hindrance. The peptide bond between proline and its preceding amino acid (Xaa-Pro) typically exists as a mixture of cis- and trans-isomers in the unfolded protein. Cis–trans isomerization of Xaa-Pro peptide bonds are infrequent, but still occur in folded proteins. Therefore, the effects of the cis–trans isomerization equilibrium in both unfolded and folded states should be taken into account when estimating the stability contribution of a specific proline residue. In order to study the stability contribution of the four proline residues to the hyperthermophilic protein Ssh10b, in this work, we expressed and purified a series of Pro→Ala mutants of Ssh10b, and performed correlative unfolding experiments in detail. We proposed a new unfolding model including proline isomerization. The model predicts that the contribution of a proline residue to protein stability is associated with the thermodynamic equilibrium between cis- and trans-isomers both in the unfolded and folded states, agreeing well with the experimental results.     

The DBA.2 mouse is susceptible to disease following infection with a broad, but limited, range of influenza A and B viruses

We assessed the relative susceptibilities to disease of the DBA.2 and C57BL/6 mouse models upon infection with a range of influenza A and B viruses. DBA.2 mice were more susceptible to disease upon inoculation with human H1N1 influenza A virus strains, several swine influenza viruses, and influenza B viruses but were not overtly susceptible to infection with human seasonal H3N2 strains. Hemagglutination inhibition and immunoglobulin isotype profiling indicated that DBA.2 and C57BL/6 mice generate comparable humoral responses upon equivalent 50% mouse lethal dose (MLD(50)) challenges with influenza virus. Our data demonstrate the utility of DBA.2 mice for the elucidation of influenza virus pathogenicity determinants and the testing of influenza vaccines.     

The protein of a new gene,Tctex4, interacts with protein kinase CK2beta subunit and is highly expressed in mouse testis

Casein kinase 2 (CK2) is a ubiquitous, multifunctional eukaryotic serine/threonine kinase that phosphorylates an array of proteins. CK2 is a heterotetramer composed of two catalytic (alpha,alpha(')) and two regulatory (beta) subunits. CK2 plays an essential role in regulatory pathways in cell transformation and proliferation. But the role and function of the individual subunits of CK2, which are not in the holoenzyme, are not yet clear. Northern blot analysis reveals the highest CK2beta activity in mouse testicles and brain. By employing a yeast two-hybrid screen to identify the proteins that interact with CK2beta, we have isolated a cDNA clone encoding a 14-kDa protein with homology to dynein light chains and have designated it as Tctex4. CK2beta interacts specifically with Tctex4 both in a yeast two-hybrid system and in an in vitro interaction assay. Northern blot and in situ hybridization showed that Tctex4 is a novel gene that is expressed in mouse testis.     

The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain

Efficient protein synthesis in bacteria requires initiation factor 2 (IF2), elongation factors Tu (EF-Tu) and G (EF-G), and release factor 3 (RF3), each of which catalyzes a major step of translation in a GTP-dependent fashion. Previous reports have suggested that recruitment of factors to the ribosome and subsequent GTP hydrolysis involve the dimeric protein L12, which forms a flexible "stalk" on the ribosome. Using heteronuclear NMR spectroscopy we demonstrate that L12 binds directly to the factors IF2, EF-Tu, EF-G, and RF3 from Escherichia coli, and map the region of L12 involved in these interactions. Factor-dependent chemical shift changes show that all four factors bind to the same region of the C-terminal domain of L12. This region includes three strictly conserved residues, K70, L80, and E82, and a set of highly conserved residues, including V66, A67, V68 and G79. Upon factor binding, all NMR signals from the C-terminal domain become broadened beyond detection, while those from the N-terminal domain are virtually unaffected, implying that the C-terminal domain binds to the factor, while the N-terminal domain dimer retains its rotational freedom mediated by the flexible hinge between the two domains. Factor-dependent variations in linewidths further reveal that L12 binds to each factor with a dissociation constant in the millimolar range in solution. These results indicate that the L12-factor complexes will be highly populated on the ribosome, because of the high local concentration of ribosome-bound factor with respect to L12.     

C-terminal domain of p16(INK4a) is adequate in inducing cell cycle arrest, growth inhibition and CDK4/6 interaction similar to the full length protein in HT-1080 fibrosarcoma cells

The tumor suppressor p16(INK4a) has earned widespread attention in cancer studies since its discovery as an inhibitor of cyclin-dependent kinases (CDKs) 4/6. Structurally, it consists of four complete ankyrin repeats, believed to be involved in CDK4 interaction. According to the previous disparities concerning the importance of domains and inactivating mutations in p16, we aimed to search for the domain possessing the functional properties of the full length protein. Upon our in silico screening analyses followed by experimental assessments, we have identified the novel minimum functional domain of p16 to be the C-terminal half including ankyrin repeats III, IV and the C-terminal flanking region accompanied by loops 2 and 3. Transfection of this truncated form into HT-1080 human fibrosarcoma cells, lacking endogenous p16, revealed that it is able to inhibit cell growth and proliferation equivalent to p16(INK4a). The functional analysis showed that this fragment like p16 can interact with CDK4/6, block the entry into S phase of the cell cycle and suppress growth as indicated by colony formation assay. Identification of p16 minimum functional domain can be of benefit to the future peptidomimetic drug design as well as gene transfer for cancer therapy.     

The Drosophila IgC2 domain protein Friend-of-Echinoid,a paralogue of Echinoid,limits the number of sensory organ precursors in the wing disc and interacts with the Notch signaling pathway

The Notch signaling pathway is critical in cell fate specification throughout development. In the developing wing disc, single sensory organ precursors (SOPs) are selected from proneural clusters via a process of lateral inhibition mediated by the Notch signaling pathway. The epidermal growth factor receptor (EGFR) pathway has also been implicated in SOP formation. Here, we describe the Drosophila melanogaster gene friend of echinoid (fred), a paralogue of echinoid (ed), a gene recently identified as a negative regulator of the EGFR pathway. fred function was examined in transgenic flies by using inducible RNA interference (RNAi). Suppression of fred in developing wing discs results in specification of ectopic SOPs, additional microchaeta, and cell death. In eye-antennal discs, fred suppression causes a rough eye phenotype. These phenotypes are suppressed by overexpression of Notch, Suppressor of Hairless [Su(H)], and Enhancer of split m7. In contrast, overexpression of Hairless, a negative regulator of the Notch pathway, and decreased Su(H) activity enhance these phenotypes. Thus, fred acts in close concert with the Notch signaling pathway. Dosage-sensitive genetic interaction also suggests a close relationship between fred and ed.     

csmA,a gene encoding a class V chitin synthase with a myosin motor-like domain of Aspergillus nidulans,is translated as a single polypeptide and regulated in response to osmotic conditions

The csmA gene of Aspergillus nidulans encodes a polypeptide that consists of an N-terminal myosin motor-like domain and a C-terminal chitin synthase domain. csmA null mutants showed marked abnormalities in polarized growth, hyphal wall integrity, and conidiophore development. Furthermore, the growth of the csmA null mutants was sensitive to low osmotic conditions. In an effort to investigate the intracellular behavior of the csmA product (CsmA) and the regulation of its production, we constructed strains that produced CsmA tagged with nine repeats of the hemagglutinin A (HA) epitope at its COOH terminus (CsmA-HA) instead of CsmA. Western blot analysis with anti-HA antibody showed that the entire coding region of csmA was translated as a single polypeptide with an approximate molecular mass of 210kDa. CsmA-HA was produced during vegetative growth; however, its yield was significantly reduced under high osmotic conditions, suggesting that the role of CsmA in growth and morphogenesis is particularly important under low osmotic conditions.