Protein splicing: its discovery and structural insight into novel chemical mechanisms

Protein splicing is a posttranslational cellular process, in which an intervening protein sequence (intein) is self-catalytically excised out from a nascent protein precursor and the two flanking sequences (N- and C-exteins) are ligated to produce two mature enzymes. This unique reaction was first discovered from studies of the structure and expression of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and yet comprises two independent genetic information for Vma1p (a catalytic 70-kDa subunit of the vacuolar H+-ATPase) and VDE (a 50-kDa DNA endonuclease) as an in-frame spliced insert in the gene. Subsequent studies have demonstrated that protein splicing is not unique for the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its precise reaction mechanisms from a viewpoint of structure-directed chemistry, a series of crystal structural studies has been carried out with the use of splicing-inactive and slowly spliceable precursors of VMA1 recombinants. One precursor structure revealed that the N-terminal junction of the introduced extein polypeptide forms an intermediate containing a five-membered thiazolidine ring. The other precursor structures showed spliced products with a linkage between the N- and C-extein segments. This article summarizes biochemical and structural studies on a self-catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation.     

Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells

Interactions between B and T cells are essential for most antibody responses, but the dynamics of these interactions are poorly understood. By two-photon microscopy of intact lymph nodes, we show that upon exposure to antigen, B cells migrate with directional preference toward the B-zone–T-zone boundary in a CCR7-dependent manner, through a region that exhibits a CCR7-ligand gradient. Initially the B cells show reduced motility, but after 1 d, motility is increased to approximately 9 μm/min. Antigen-engaged B cells pair with antigen-specific helper T cells for 10 to more than 60 min, whereas non-antigen-specific interactions last less than 10 min. B cell–T cell conjugates are highly dynamic and migrate extensively, being led by B cells. B cells occasionally contact more than one T cell, whereas T cells are strictly monogamous in their interactions. These findings provide evidence of lymphocyte chemotaxis in vivo, and they begin to define the spatiotemporal cellular dynamics associated with T cell–dependent antibody responses.     

Simple and rapid determination of metabolite content in plant cell culture medium using an FT-IR/ATR method

A simple, rapid and accurate mid-infrared (MIR) spectroscopic method for simultaneously determining the product (ethanol) content and the nutrient (sugar) content in plant-cell culture media was developed using a Fourier transform infrared (FT-IR) spectrometer equipped with an attenuated total reflectance (ATR) accessory. We assessed the potential of this method by comparing it to a high-performance liquid chromatography (HPLC) method, and using the developed method to measure the ethanol and sugar contents simultaneously in liquid culture media with rice and tabacum cell suspensions, respectively. The experimental results suggest that the sugar consumption and ethanol production behaviors of the plant cell suspensions can be non-destructively and simultaneously monitored using the developed method. Furthermore, the spectroscopic method provided in this study could be developed into a technique that could be used to analyze the overall kinetics of the metabolism of the plant cell suspensions.     

MIR spectroscopic analysis on sugar metabolic and ethanol productive kinetics of suspension TBY-2 and rice cells pre-cultured in various media

The influence of sugars in pre-cultivation media suspended plant cells on the kinetics of the sugar uptake and the ethanol production was studied by mid-infrared spectroscopy using a Fourier transform infrared spectrometer (FT-IR) equipped with an attenuate total reflection accessory (ATR). We performed the plant cell cultivation with Nicotiana tabacum cv. Bright Yellow No.2 (TBY-2) cells and Oryza sativa L., Japonica, cv. Nipponbare (rice) cells, respectively, in pre-culture and culture media, which had various types of glucose, fructose, sucrose or glucose–fructose mixtures. The results confirmed the kinetic differences between the TBY-2 cells and rice cells. These results suggested that the TBY-2 cells consumed sugar before growth and the rice cells consumed sugar after growth, moreover, the ethanol content increased just after cell growth was activated based on the non-dimensional cultivation time for the cell growth behavior.     

Dipeptidyl peptidase IV in tumor progression

Dipeptidyl peptidase IV (DPPIV) is a 110-kDa glycoprotein with ubiquitous expression. Several recent studies have shown that DPPIV affects tumor progression in several human malignancies. We found that ovarian carcinoma cell lines with higher DPPIV expression showed less invasive potential. Furthermore, introduction of DPPIV cDNA into SKOV3 cells (SKDPIV), derived from serous cystadenocarcinoma showing little DPPIV expression, caused a significant decrease in both migration and invasive potential. In addition, nude mice inoculated with SKDPIV cells showed significantly less peritoneal dissemination and longer survival time than those inoculated with parental or vector-transfected cells. We further examined the mechanisms of anti-invasive ability of DPPIV. The expression of E-cadherin was positively correlated with DPPIV expression among five independent ovarian carcinoma cell lines. The SKDPIV cells showed enhanced expression of E-cadherin with a cellular morphological change from a fibroblastic and motile phenotype to an epithelial phenotype compared to parental and MOCK cells. In addition, matrix metalloproteinase 2 (MMP-2) and membrane type 1 matrix metalloprotease (MT1-MMP), which are important markers associated with invasive and metastatic potential, were remarkably reduced in SKDPIV cells. In contrast, tissue inhibitors of matrix metalloproteinases (TIMPs) were enhanced by DPPIV transfection. These findings imply that DPPIV may functionally suppress peritoneal dissemination and progression of ovarian carcinoma by regulating the expression levels of several molecules associated with carcinoma cell invasion and progression.     

Aberrant glycosylation of alpha-dystroglycan causes defective binding of laminin in the muscle of chicken muscular dystrophy

Dystroglycan is a central component of dystrophin-glycoprotein complex that links extracellular matrix and cytoskeleton in skeletal muscle. Although dystrophic chicken is well established as an animal model of human muscular dystrophy, the pathomechanism leading to muscular degeneration remains unknown. We show here that glycosylation and laminin-binding activity of alpha-dystroglycan (alpha-DG) are defective in dystrophic chicken. Extensive glycan structural analysis reveals that Galbeta1-3GalNAc and GalNAc residues are increased while Siaalpha2-3Gal structure is reduced in alpha-DG of dystrophic chicken. These results implicate aberrant glycosylation of alpha-DG in the pathogenesis of muscular degeneration in this model animal of muscular dystrophy.     

Structural characterization of the proximal and distal histidine environment of cytoglobin and neuroglobin

Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (nu(Fe-His)) bands at 229 and 221 cm(-1), respectively. No time-dependent shift in the nu(Fe-His) band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (nu(Fe-CO), nu(C-O), delta(Fe-C-O)) and (nu(Fe-NO), nu(N-O), delta(Fe-N-O)) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.     

ADAMs, a disintegrin and metalloproteinases, mediate shedding of oxytocinase

Placental leucine aminopeptidase (P-LAP), a type-II transmembrane protease responsible for oxytocin degradation during pregnancy, is converted to a soluble form through proteolytic cleavage. The goal of this study was to determine the nature of the P-LAP secretase activity. The hydroxamic acid-based metalloprotease inhibitors GM6001 and ONO-4817 as well as the TNF-alpha protease inhibitor-2 (TAPI-2) reduced P-LAP release, while tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2, which are matrix metalloproteinase inhibitors, had no effect on P-LAP release in Chinese hamster ovary (CHO) cells stably overexpressing P-LAP, thus indicating possible involvement of ADAM (a disintegrin and metalloproteinase) members in P-LAP shedding. Furthermore, overexpression of ADAM9 and ADAM12 increased P-LAP release in P-LAP-CHO transfectants. Immunohistochemical analysis in human placenta demonstrated strong expression of ADAM12 in syncytiotrophoblasts, while little expression of ADAM9 was detected throughout the placenta. Our results suggest ADAM members, at least including ADAM12, are involved in P-LAP shedding in human placenta.     

Cardiolipin induces premature senescence in normal human fibroblasts

Lipids seem to have various roles in cellular senescence. We found that cardiolipin very sensitively inhibits growth of normal human fibroblasts, whereas other phospholipids do not at 100 times higher concentrations. Growth arrested cells showed morphology similar to those of normally senesced cells and strongly induced senescence-associated beta-galactosidase. Senescence markers such as the p21(waf1/sdi-1), fibronectin, and collagenase-I genes were significantly upregulated by cardiolipin. In addition, caldiolipin significantly increased in normally senesced human fibroblasts leaving other phospholipids unaltered. These results suggest that accumulation of cardiolipin is one of the causes for replicative senescence.