Identification of a SNARE protein required for vacuolar protein transport in Schizosaccharomyces pombe

Intracellular vesicle trafficking is mediated by a set of SNARE proteins in eukaryotic cells. Several SNARE proteins are required for vacuolar protein transport and vacuolar biogenesis in Saccharomyces cerevisiae. A search of the Schizosaccharomyces pombe genome database revealed a total of 17 SNARE-related genes. Although no homologs of Vam3p, Nyv1p, and Vam7p have been found in S. pombe, we identified one SNARE-like protein that is homologous to S. cerevisiae Pep12p. However, the disruptants transport vacuolar hydrolase CPY (SpCPY) to the vacuole normally, suggesting that the Pep12 homolog is not required for vacuolar protein transport in S. pombe cells. To identify the SNARE protein(s) involved in Golgi-to-vacuole protein transport, we have deleted four SNARE homolog genes in S. pombe. SpCPY was significantly missorted to the cell surface on deletion of one of the SNARE proteins, Fsv1p (SPAC6F12.03c), with no apparent S. cerevisiae ortholog. In addition, sporulation, endocytosis, and in vivo vacuolar fusion appear to be normal in fsv1Delta cells. These results showed that Fsv1p is mainly involved in vesicle-mediated protein transport between the Golgi and vacuole in S. pombe cells.     

Distinct sites regulating grayanotoxin binding and unbinding to D4S6 of Na(v)1.4 sodium channel as revealed by improved estimation of toxin sensitivity

Grayanotoxin (GTX) exerts selective effects on voltage-dependent sodium channels by eliminating fast sodium inactivation and causing a hyperpolarizing shift in voltage dependence of channel activation. In this study, we adopted a newly developed protocol that provides independent estimates of the binding and unbinding rate constants of GTX (k(on) and k(off)) to GTX sites on the sodium channel protein, important in the molecular analysis of channel modification. Novel GTX sites were determined in D2S6 (Asn-784) and D3S6 (Ser-1276) by means of site-directed mutagenesis; the results suggested that the GTX receptor consists of the S6 transmembrane segments of four homologous domains facing the ion-conducting pore. We systematically introduced at two sites in D4S6 (Na(v)1.4-Phe-1579 and Na(v)1.4-Tyr-1586) amino acid substituents with residues containing hydrophobic, aromatic, charged, or polar groups. Generally, substitutions at Phe-1579 increased both k(on) and k(off), resulting in no prominent change in dissociation constant (K(d)). It seems that the smaller the molecular size of the residue at Na(v)1.4-Phe-1579, the larger the rates of k(on) and k(off), indicating that this site acts as a gate regulating access of toxin molecules to a receptor site. Substitutions at Tyr-1586 selectively increased k(off) but had virtually no effect on k(on), thus causing a drastic increase in K(d). At position Tyr-1586, a hydrophobic or aromatic amino acid side chain was required to maintain normal sensitivity to GTX. These results suggest that the residue at position Tyr-1586 has a more critical role in mediating GTX binding than the one at position Phe-1579. Here, we propose that the affinity of GTX to Na(v)1.4 sodium channels might be regulated by two residues (Phe and Tyr) at positions Phe-1579 and Tyr-1586, which, respectively, control access and binding of GTX to its receptor.     

The N-terminal region of the transmembrane domain of human erythrocyte band 3. Residues critical for membrane insertion and transport activity

We studied the role of the N-terminal region of the transmembrane domain of the human erythrocyte anion exchanger (band 3; residues 361-408) in the insertion, folding, and assembly of the first transmembrane span (TM1) to give rise to a transport-active molecule. We focused on the sequence around the 9-amino acid region deleted in Southeast Asian ovalocytosis (Ala-400 to Ala-408), which gives rise to nonfunctional band 3, and also on the portion of the protein N-terminal to the transmembrane domain (amino acids 361-396). We examined the effects of mutations in these regions on endoplasmic reticulum insertion (using cell-free translation), chloride transport, and cell-surface movement in Xenopus oocytes. We found that the hydrophobic length of TM1 was critical for membrane insertion and that formation of a transport-active structure also depended on the presence of specific amino acid sequences in TM1. Deletions of 2 or 3 amino acids including Pro-403 retained transport activity provided that a polar residue was located 2 or 3 amino acids on the C-terminal side of Asp-399. Finally, deletion of the cytoplasmic surface sequence G(381)LVRD abolished chloride transport, but not surface expression, indicating that this sequence makes an essential structural contribution to the anion transport site of band 3.     

Gene therapy for diabetes mellitus

There are diverse strategies for gene therapy of diabetes mellitus. Prevention of beta-cell autoimmunity is a specific gene therapy for prevention of type 1 (insulin-dependent) diabetes in a preclinical stage, whereas improvement in insulin sensitivity of peripheral tissues is a specific gene therapy for type 2 (non-insulin-dependent) diabetes. Suppression of beta-cell apoptosis, recovery from insulin deficiency, and relief of diabetic complications are common therapeutic approaches to both types of diabetes. Several approaches to insulin replacement by gene therapy are currently employed: 1) stimulation of beta-cell growth, 2) induction of beta-cell differentiation and regeneration, 3) genetic engineering of non-beta cells to produce insulin, and 4) transplantation of engineered islets or beta cells. In type 1 diabetes, the therapeutic effect of beta-cell proliferation and regeneration is limited as long as the autoimmune destruction of beta cells continues. Therefore, the utilization of engineered non-beta cells free from autoimmunity and islet transplantation with immunological barriers are considered potential therapies for type 1 diabetes. Proliferation of the patients' own beta cells and differentiation of the patients' own non-beta cells to beta cells are desirable strategies for gene therapy of type 2 diabetes because immunological problems can be circumvented. At present, however, these strategies are technically difficult, and transplantation of engineered beta cells or islets with immunological barriers is also a potential gene therapy for type 2 diabetes.     

Adjustment function among antioxidant substances in acatalasemic mouse brain and its enhancement by low-dose X-ray irradiation

The catalase activities in blood and organs of the acatalasemic (C3H/AnLCsbCsb) mouse of the C3H strain are lower than those of the normal (C3H/AnLCsaCsa) mouse. We conducted a study to examine changes in the activities of antioxidant enzymes, such as catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX), the total gluathione content, and the lipid peroxide level in the brain, which is more sensitive to oxidative stress than other organs, at 3, 6, or 24 hr following X-ray irradiation at doses of 0.25, 0.5, or 5.0 Gy to the acatalasemic and the normal mice. No significant change in the lipid peroxide level in the acatalasemic mouse brain was seen under non-irradiation conditions. However, the acatalasemic mouse brain was more damaged than the normal mouse brain by excessive oxygen stress, such as a high-dose (5.0 Gy) X-ray. On the other hand, we found that, unlike 5.0 Gy X-ray, a relatively low-dose (0.5 Gy) irradiation specifically increased the activities of both catalase and GPX in the acatalasemic mouse brain making the activities closer to those in the normal mouse brain. These findings may indicate that the free radical reaction induced by the lack of catalase is more properly neutralized by low dose irradiation.     

Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.     

T cells of atomic bomb survivors respond poorly to stimulation by Staphylococcus aureus toxins in vitro: does this stem from their peripheral lymphocyte populations having a diminished naïve CD4 T-cell content?

We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer na?ve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (na?ve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (na?ve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their na?ve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.     

Activation of the lectin complement pathway by H-ficolin (Hakata antigen)

Ficolins are a group of proteins which consist of a collagen-like domain and a fibrinogen-like domain. In human serum, there are two types of ficolins named L-ficolin/P35 and H-ficolin (Hakata Ag), both of which have lectin activity. We recently demonstrated that L-ficolin/P35 is associated with mannose-binding lectin (MBL)-associated serine proteases (MASP) 1 and 2 and small MBL-associated protein (sMAP), and that the complex activates the lectin pathway. In this study, we report the characterization of H-ficolin in terms of its ability to activate complement. Western blotting analysis showed the presence of MASP-1, MASP-2, MASP-3, and sMAP in H-ficolin preparations isolated from Cohn Fraction III. The MASPs in the preparations had proteolytic activities against C4, C2, and C3 in the fluid phase. When H-ficolin preparations were bound to anti-H-ficolin Ab which had been coated on ELISA plates, they activated C4, although no C4 activation was noted when anti-MBL and anti-L-ficolin/P35 were used. H-ficolin binds to PSA, a polysaccharide produced by Aerococcus viridans. C4 was activated by H-ficolin preparations bound to PSA which had been coated on ELISA plates. These results indicate that H-ficolin is a second ficolin which is associated with MASPs and sMAP, and which activates the lectin pathway.