Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4.

Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 Å resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site.

Comparison of the structure and activity of thioredoxin 2 and thioredoxin 1 from Acinetobacter baumannii.

Thioredoxin (Trx) is essential in a redox-control system, with many bacteria containing two Trxs: Trx1 and Trx2. Due to a Trx system's critical function, Trxs are targets for novel antibiotics. Here, a 1.20 Šhigh-resolution structure of Trx2 from Acinetobacter baumannii (abTrx2), an antibiotic resistant pathogenic superbug, is elucidated. By comparing Trx1 and Trx2, it is revealed that the two Trxs possess similar activity, although Trx2 contains an additional N-terminal zinc-finger domain and exhibits more flexible properties in solution. Finally, it is shown that the Trx2 zinc-finger domain might be rotatable and that proper zinc coordination at the zinc-finger domain is critical to abTrx2 activity. This study enhances understanding of the Trx system and will facilitate the design of novel antibiotics.

Generation of human TMEM16F-specific affibodies using purified TMEM16F.

Introduction: TMEM16 family proteins are involved in a variety of functions, including ion transport, phospholipid scrambling, and the regulation of membrane proteins. Among them, TMEM16F has dual functions as a phospholipid scramblase and a nonselective ion channel. TMEM16F is widely expressed and functions in platelet activation during blood clotting, bone formation, and T cell activation. Despite the functional importance of TMEM16F, the modulators of TMEM16F function have not been sufficiently studied. Method: In this study, we generated TMEM16F-specific affibodies by performing phage display with brain-specific TMEM16F (hTMEM16F) variant 1 purified from GnTi- cells expressing this variant in the presence of digitonin as a detergent. Purified human TMEM16F protein, which was proficient in transporting phospholipids in a Ca2+-dependent manner in proteoliposomes, was coated onto plates and then the phage library was added to fish out TMEM16F-binding affibodies. For the validation of interaction between affibodies and TMEM16F proteins, ELISA, bio-layer interferometry, and size exclusion chromatography were conducted. Results and Discussion: As a result, the full sequences of 38 candidates were acquired from 98 binding candidates. Then, we selected 10 candidates and purified seven of them from E. coli expressing these candidates. Using various assays, we confirmed that two affibodies bound to human TMEM16F with high affinity. These affibodies can be useful for therapeutical and diagnostic applications of TMEM16F-related cancer and neurodegenerative diseases. Future studies will be required to investigate the effects of these affibodies on TMEM16F function.

Enhancement of the immunosuppressive effect of human adipose tissue-derived mesenchymal stromal cells through HLA-G1 expression.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) from several tissues have immunomodulatory properties that involve various immunosuppressive molecules. An example is human leukocyte antigen (HLA)-G, a non-classical major histocompatibility complex (MHC) class I molecule that induces tolerance via interactions with inhibitory receptors present on major immune effector cells. Recently, the molecular mechanisms that regulate MSC-mediated immunosuppression have come under investigation. Our goal was to determine whether HLA-G plays a crucial role in immunosuppression and whether human adipose tissure (hAT) MSC can be used as a tool for biologic immunosuppression with HLA-G in transplantation. METHODS: MSC were characterized by fluorescence-activated cell sorting (FACS) analysis, reverse transcriptase (RT)-polymerase chain reaction (PCR) and staining for differentiation. The immunogenicity and immunomodulatory effects of MSC were monitored by peripheral blood mononuclear cell (PBMC) proliferation assay with or without phytohemagglutinin (PHA) stimulation. Stable expression of HLA-G1 in MSC was done using a lentiviral system. Results. MSC from different tissues had similar morphology, immunophenotypic characters and differentiation potential. We also found that the immunosuppressive effect of MSC was monitored along with their endogenous HLA-G mRNA and protein levels. Stable expression of HLA-G1 appeared to enhance the immunosuppressive effect of hAT MSC, and the function of HLA-G1 was significantly decreased by HLA-G antagonistic antibody in PBMC proliferation assays. CONCLUSIONS: Although the HLA-G molecule is not the sole factor for MSC-mediated immunosuppression, our data provide evidence that HLA-G plays an important role in immunosuppression and that hAT MSC can be used as a tool for biologic immunosuppression during transplantation procedures.