TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.     

Functional characterization of extracellular chitinase encoded by the YlCTS1 gene in a dimorphic yeast Yarrowia lipolytica

The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by O-mannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.     

Apoptosis signal-regulating kinase-1 aggravates ROS-mediated striatal degeneration in 3-nitropropionic acid-infused mice

Apoptosis signal-regulating kinase-1 (ASK1), an early signaling element in the cell death pathway, has been suggested to participate in the pathology of neurodegenerative diseases, which may be associated with environmental factors that impact the diseases. Although it is not entirely elucidated, 3-nitropropionic acid (3-NP) provokes mitochondrial dysfunction and selectively forms striatal lesions similar to those found in Huntington’s disease. The current study investigated whether ASK1 is involved in striatal pathology following chronic systemic infusion of 3-NP. The results show that ASK1 acts as a primary mediator of there active oxygen species (ROS) cell death signal cascade in the 3-NP-damaged striatal region by disrupting the positive feedback cycle. In 3-NP-infused striatal lesions, ROS increased ASK1. Superoxide dismutase transgenic (SOD-tg) mice reduced ASK1by scavenging ROS, and reduction of ASK1leads to a reduction in cell death. However, ASK1 down-regulation in 3-NP infusion mice also decreased striatal cell death without scavenging ROS. In contrast decreasing cell death by si-ASK1 treatment along with 3-NP in both SOD tg and wild-type mice (wt), cell death rebounded when ASK1 peptide was added to SOD tg mice. The present study suggests that ROS-inducing ASK1 may be an important step in the pathogenesis of 3-NP infused striatal lesions in murine brains.     

Diversity of Plant Methionine Sulfoxide Reductases B and Evolution of a Form Specific for Free Methionine Sulfoxide

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological conditions. Organisms evolved two distinct methionine sulfoxide reductase families (MSRA & MSRB) to repair oxidized methionine residues. We found that 5 MSRB genes exist in the soybean genome, including GmMSRB1 and two segmentally duplicated gene pairs (GmMSRB2 and GmMSRB5, GmMSRB3 and GmMSRB4). GmMSRB2 and GmMSRB4 proteins showed MSRB activity toward protein-based MetO with either DTT or thioredoxin (TRX) as reductants, whereas GmMSRB1 was active only with DTT. GmMSRB2 had a typical MSRB mechanism with Cys121 and Cys 68 as catalytic and resolving residues, respectively. Surprisingly, this enzyme also possessed the MSRB activity toward free Met-R-O with kinetic parameters similar to those reported for fRMSR from Escherichia coli, an enzyme specific for free Met-R-O. Overexpression of GmMSRB2 or GmMSRB4 in the yeast cytosol supported the growth of the triple MSRA/MSRB/fRMSR (Δ3MSRs) mutant on MetO and protected cells against H₂O₂-induced stress. Taken together, our data reveal an unexpected diversity of MSRBs in plants and indicate that, in contrast to mammals that cannot reduce free Met-R-O and microorganisms that use fRMSR for this purpose, plants evolved MSRBs for the reduction of both free and protein-based MetO.     

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.     

Development of a risk scoring system for patients with papillary thyroid cancer

As the importance of personalized therapeutics in aggressive papillary thyroid cancer (PTC) increases, accurate risk stratification is required. To develop a novel prognostic scoring system for patients with PTC (n = 455), we used mRNA expression and clinical data from The Cancer Genome Atlas. We performed variable selection using Network‐Regularized high‐dimensional Cox‐regression with gene network from pathway databases. The risk score was calculated using a linear combination of regression coefficients and mRNA expressions. The risk score and clinical variables were assessed by several survival analyses. The risk score showed high discriminatory power for the prediction of event‐free survival as well as the presence of metastasis. In multivariate analysis, the risk score and presence of metastasis were significant risk factors among the clinical variables that were examined together. In the current study, we developed a risk scoring system that will help to identify suitable therapeutic options for PTC.