The SV40 Capsid Is Stabilized by a Conserved Pentapeptide Hinge of the Major Capsid Protein VP1

The simian virus 40 (SV40) outer shell is composed of 72 pentamers of VP1. The core of the VP1 monomer is a β-barrel with jelly-roll topology and extending N- and C-terminal arms. A pentapeptide hinge, KNPYP, tethers the C-arm to the VP1 β-barrel core. The five C-arms that extend from each pentamer insert into the neighbouring pentamers, tying them together through different types of interactions. In the mature virion, this element adopts either of six conformations according to their location in the capsid. We found that the hinge is conserved among 16 members of the Polyomaviridae, attesting to its importance in capsid assembly and/or structure. We have used site-directed mutagenesis to gain an understanding into the structural requirements of this element: Y299 was changed to A, F, and T, and P300 to A and G. The mutants showed reduction in viability to varying degrees. Unexpectedly, assembly was reduced only to a small extent. However, the data showed that the mutants were highly unstable. The largest effect was observed for mutations of P300, indicating a role of the proline in the virion structure. P300G was more unstable than P300A, indicating a requirement for rigidity of the pentapeptide hinge. Y299T and Y299A were more defective in viability than Y299F, highlighting the importance of an aromatic ring at this position. Structural inspection showed that this aromatic ring contacts C-arms of neighbouring pentamers. Computational modelling predicted loss of stability of the Y mutants in concordance with the experimental results. This study provides insights into the structural details of the pentapeptide hinge that are responsible for capsid stability.     

Loss of Prolyl Carboxypeptidase in Two-Kidney,One-Clip Goldblatt Hypertensive Mice

It is well documented that angiotensin (Ang) II contributes to kidney disease progression. The protease prolyl carboxypeptidase (PRCP) is highly expressed in the kidney and may be renoprotective by degrading Ang II to Ang-(1-7). The aim of the study was to investigate whether renal PRCP protein expression and activity are altered in two-kidney, one-clip (2K1C) Goldblatt hypertensive mice. Left renal artery was constricted by using 0.12 mm silver clips. Blood pressure was measured using telemetry over the eleven weeks of study period and revealed an immediate increase in 2K1C animals during the first week of clip placement which was followed by a gradual decrease to baseline blood pressure. Similarly, urinary albumin excretion was significantly increased one week after 2K1C and returned to baseline levels during the following weeks. At 2 weeks and at the end of the study, renal pathologies were exacerbated in the 2K1C model as revealed by a significant increase in mesangial expansion and renal fibrosis. Renal PRCP expression and activity were significantly reduced in clipped kidneys. Immunofluorescence revealed the loss of renal tubular PRCP but not glomerular PRCP. In contrast, expression of prolyl endopeptidase, another enzyme capable of converting Ang II into Ang-(1-7), was not affected, while angiotensin converting enzyme was elevated in unclipped kidneys and renin was increased in clipped kidneys. Results suggest that PRCP is suppressed in 2K1C and that this downregulation may attenuate renoprotective effects via impaired Ang II degradation by PRCP.     

The phosphatidylinositol‐transfer protein Nir2 binds phosphatidic acid and positively regulates phosphoinositide signalling

Phosphatidic acid (PA) and phosphoinositides are metabolically interconverted lipid second messengers that have central roles in many growth factor (GF)‐stimulated signalling pathways. Yet, little is known about the mechanisms that coordinate their production and downstream signalling. Here we show that the phosphatidylinositol (PI)‐transfer protein Nir2 translocates from the Golgi complex to the plasma membrane in response to GF stimulation. This translocation is triggered by PA formation and is mediated by its C‐terminal region that binds PA in vitro. We further show that depletion of Nir2 substantially reduces the PI(4,5)P2 levels at the plasma membrane and concomitantly GF‐stimulated PI(3,4,5)P3 production. Finally, we show that Nir2 positively regulates the MAPK and PI3K/AKT pathways. We propose that Nir2 through its PA‐binding capability and PI‐transfer activity can couple PA to phosphoinositide signalling, and possibly coordinates their local lipid metabolism and downstream signalling.     

Adhesion of Vibrio cholerae to granular starches

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. Cholera can become epidemic and deadly without adequate medical care. Appropriate rehydration therapy can reduce the mortality rate from as much as 50% of the affected individuals to <1%. Thus, oral rehydration therapy (ORT) is an important measure in the treatment of this disease. To further reduce the symptoms associated with cholera, improvements in oral rehydration solution (ORS) by starch incorporation were suggested. Here, we report that V. cholerae adheres to starch granules incorporated in ORS. Adhesion of 98% of the cells was observed within 2 min when cornstarch granules were used. Other starches showed varied adhesion rates, indicating that starch source and composition play an important role in the interaction of V. cholerae and starch granules. Sugars metabolized by V. cholerae showed a repressive effect on the adhesion process. The possible mechanisms involved are discussed. Comparing V. cholerae adhesion with the adhesion of other pathogens suggests the involvement of starch degradation capabilities. This adhesion to granular starch can be used to improve ORT.     

C6ORF66 is an assembly factor of mitochondrial complex I

Homozygosity mapping was performed in five patients from a consanguineous family who presented with infantile mitochondrial encephalomyopathy attributed to isolated NADH:ubiquinone oxidoreductase (complex I) deficiency. This resulted in the identification of a missense mutation in a conserved residue of the C6ORF66 gene, which encodes a 20.2 kDa mitochondrial protein. The mutation was also detected in a patient who presented with antenatal cardiomyopathy. In muscle of two patients, the levels of the C6ORF66 protein and of the fully assembled complex I were markedly reduced. Transfection of the patients' fibroblasts with wild-type C6ORF66 cDNA restored complex I activity. These data suggest that C6ORF66 is an assembly factor of complex I. Interestingly, the C6ORF66 gene product was previously shown to promote breast cancer cell invasiveness.     

Possible regulation of the Salmonella typhimurium histidine operon by adenosine triphosphate phosphoribosyltransferase: large metabolic effects.

An effort to find growth conditions leading to conditional regulation of the histidine operon of Salmonella typhimurium by the allosteric first enzyme of the pathway, adenosine triphosphate phosphoribosyltransferase (EC 2.4.2.17), is reported. A strain deleting the enzyme, TR3343, behaved simply and predictably under all growth conditions, whereas histidine auxotrophs containing active enzyme behaved in complicated ways dependent upon the location of the histidine pathway lesion. hisE strains derepressed the operon only one-half as much as TR3343 when grown on limiting histidine and a poor carbon source, but they also grew more slowly, probably as a result of high N1-(5-phospho-beta-D-ribosyl)-adenosine triphosphate levels in the cell. hisC strains exhibited oscillatory growth behavior and oscillatory histidine operon expression when grown on intermediate concentrations of the histidine precursor histidinol. This behavior probably was caused by synergistic in-phase variations in the histidine, purine nucleotide, and ppGpp pools of the cell. All of the growth and histidine operon expression effects associated with the presence of adenosine triphosphate phosphoribosyltransferase could be assigned to metabolic perturbation of the cell caused by unregulated enzymatic activity.     

Cooperative interactions at the SLP‐76 complex are critical for actin polymerization

T‐cell antigen receptor (TCR) engagement induces formation of multi‐protein signalling complexes essential for regulating T‐cell functions. Generation of a complex of SLP‐76, Nck and VAV1 is crucial for regulation of the actin machinery. We define the composition, stoichiometry and specificity of interactions in the SLP‐76, Nck and VAV1 complex. Our data reveal that this complex can contain one SLP‐76 molecule, two Nck and two VAV1 molecules. A direct interaction between Nck and VAV1 is mediated by binding between the C‐terminal SH3 domain of Nck and the VAV1 N‐terminal SH3 domain. Disruption of the VAV1:Nck interaction deleteriously affected actin polymerization. These novel findings shed new light on the mechanism of actin polymerization after T‐cell activation.