Translational control of SEPT9 isoforms is perturbed in disease

A common feature of the mammalian septin gene family is complex genomic architecture with multiple alternate splice variants. Septin 9 has 18 distinct transcripts encoding 15 polypeptides, with two transcripts (SEPT9_v4 and v4*) encoding the same polypeptide. We have previously reported that the ratio of these distinct transcripts is altered in neoplasia, with the v4 transcript being the usual form in normal cells but v4* becoming predominant in tumours. This led us to ask what the functional differences between these two transcripts might be. The 5'-UTRs of v4 and v4* have distinct 5' ends encoded by exons 1 beta (v4) and 1 zeta and 2 (v4*) and a common 3' region and initiating ATG encoded within exon 3. Here we show that the two mRNAs are translated with different efficiencies and that cellular stress can alter this. A putative internal ribosome entry site can be identified in the common region of the v4 and v4* 5'-UTRs and translation is modulated by an upstream open-reading frame in the unique region of the v4 5'-UTR. Germline mutations in hereditary neuralgic amyotrophy (HNA) map to the region which is common to the two UTRs. These mutations dramatically enhance the translational efficiency of the v4 5'-UTR, leading to elevated SEPT9_v4 protein under hypoxic conditions. Our data provide a mechanistic insight into how the HNA mutations can alter the fine control of SEPT9_v4 protein and its regulation under physiologically relevant conditions and are consistent with the episodic and stress-induced nature of the clinical features of HNA.

Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane(1). Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses(1). The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class I-A Pi3ks (ref. 2). Mice lacking only the p85a isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants(3). Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites, We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class I-A Pi3k catalytic subunits: nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.

Interactions of 12-lipoxygenase with phospholipase A(2) isoforms following platelet activation through the glycoprotein VI collagen receptor

Recent studies implicate the collagen receptor, glycoprotein VI (GPVI) in activation of platelet 12-lipoxygenase (p12-LOX). Herein, we show that GPVI-stimulated 12-hydro(peroxy)eicosatetraenoic acid (H(P)ETE) synthesis is inhibited by palmityl trifluromethyl ketone or oleyloxyethyl phosphocholine, but not bromoenol lactone, implicating secretory and cytosolic, but not calcium-independent phospholipase A(2) (PLA(2)) isoforms. Also, following GPVI activation, 12-LOX co-immunoprecipitates with both cytosolic and secretory PLA(2), (sPLA(2)). Finally, venoms containing sPLA(2) acutely activate p12-LOX in a dose-dependent manner. This study shows that platelet 12-H(P)ETE generation utilizes arachidonate substrate from both c- and sPLA(2) and that 12-LOX functionally associates with both PLA(2) isoforms. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Regulation of transient receptor potential melastatin 8 (TRPM8) channel activity by its short isoforms.

One important mechanism of membrane ion channels regulation involves their non-functional isoforms generated by alternative splicing. However, knowledge of such isoforms for the members of transient receptor potential (TRP) superfamily of ion channels remains quite limited. This study focuses on TRPM member, TRPM8, which functions as a cold receptor in sensory neurons, but is also expressed in tissues not exposed to ambient temperatures, as well as in cancer tissues. We report the cloning from prostate cancer cells of new short-splice variants of TRPM8, termed short TRPM8a (sM8a) and short TRPM8ß (sM8ß). Our results show that both variants are in a closed configuration with the C-terminal tail of the full-size TRPM8 chan-nel, resulting in stabilization of its closed state and thus reducing both its cold sensitivity and its activity. Our findings, therefore, uncover a new mode of the regulation of TRPM8 channel by its splice variants.

Short Isoforms of the Cold Receptor TRPM8 Inhibit Channel Gating by Mimicking Heat Action Rather than Chemical Inhibitors

Transient receptor potential (TRP) channels couple various environmental factors to changes in membrane potential, calcium influx, and cell signaling. They also integrate multiple stimuli through their typically polymodal activation. Thus, although the TRPM8 channel has been extensively investigated as the major neuronal cold sensor, it is also regulated by various chemicals, as well as by several short channel isoforms. Mechanistic understanding of such complex regulation is facilitated by quantitative single-channel analysis. We have recently proposed a single-channel mechanism of TRPM8 regulation by voltage and temperature. Using this gating mechanism, we now investigate TRPM8 inhibition in cell-attached patches using HEK293 cells expressing TRPM8 alone or coexpressed with its short sM8-6 isoform. This is compared with inhibition by the chemicals N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC) and clotrimazole or by elevated temperature. We found that within the seven-state single-channel gating mechanism, inhibition of TRPM8 by short sM8-6 isoforms closely resembles inhibition by increased temperature. In contrast, inhibition by BCTC and that by clotrimazole share a different set of common features. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis

Introduction: Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic autoimmune diseases with variable clinical outcomes. We investigated whether the synovial fluid (SF) proteome could distinguish a subset of patients in whom disease extends to affect a large number of joints.

Methods: SF samples from 57 patients were obtained around time of initial diagnosis of JIA, labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression verified by immunochemical methods. Protein glycosylation status was confirmed by hydrophilic interaction liquid chromatography.

Results: A truncated isoform of vitamin D binding protein (VDBP) is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p < 0.05). Furthermore, sialylated forms of immunopurified synovial VDBP were significantly reduced in extended oligoarticular patients (p < 0.005).

Conclusion: Reduced conversion of VDBP to a macrophage activation factor may be used to stratify patients to determine risk of disease extension in JIA patients.

Identification of MET genomic amplification, protein expression and alternative splice isoforms in neuroblastomas

Crizotinib, a dual anaplastic lymphoma kinase (ALK) and mesenchymal-epithelial transition (MET) tyrosine kinase inhibitor, is currently being evaluated for the treatment of neuroblastoma. Its effects are thought to be mediated mainly via its activity against ALK. Although MET genomic/protein expression status might conceivably affect crizotinib efficacy, this issue has hitherto not received attention in neuroblastomas.

The impact of cyclin D1 mRNA isoforms, morphology and p53 in mantle cell lymphoma: p53 alterations and blastoid morphology are strong predictors of a high proliferation index.

BACKGROUND: Mantle cell lymphoma is a clinically heterogeneous disease characterized by overexpression of cyclin D1 protein. Blastoid morphology, high proliferation, and secondary genetic aberrations are markers of aggressive behavior. Expression profiling of mantle cell lymphoma revealed that predominance of the 3'UTR-deficient, short cyclin D1 mRNA isoform was associated with high cyclin D1 levels, a high "proliferation signature" and poor prognosis. DESIGN AND METHODS: Sixty-two cases of mantle cell lymphoma were analyzed for cyclin D1 mRNA isoforms and total cyclin D1 levels by real-time reverse transcriptase polymerase chain reaction, and TP53 alterations were assessed by immunohistochemistry and molecular analysis. Results were correlated with proliferation index and clinical outcome. RESULTS: Predominance of the short cyclin D1 mRNA was found in 14 (23%) samples, including four with complete loss of the standard transcript. TP53 alterations were found in 15 (24%) cases. Predominance of 3'UTR-deficient mRNA was significantly associated with high cyclin D1 mRNA levels (P=0.009) and more commonly found in blastoid mantle cell lymphoma (5/11, P=0.060) and cases with a proliferation index of >20% (P=0.026). Both blastoid morphology (11/11, P<0.001) and TP53 alterations (15/15, P<0.001) were significantly correlated with a high proliferation index. A proliferation index of 10% was determined to be a significant threshold for survival in multivariate analysis (P=0.01). CONCLUSIONS: TP53 alterations are strongly associated with a high proliferation index and aggressive behavior in mantle cell lymphoma. Predominance of the 3'UTR-deficient transcript correlates with higher cyclin D1 levels and may be a secondary contributing factor to high proliferation, but failed to reach prognostic significance in this study.

Expression of cyclooxygenase isoforms in equine gastric ulcers

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Two isoforms of anti-lipopolysaccharide factors identified and characterized from the hemocytes of portunid crabs, Portunus pelagicus and Scylla tranquebarica

Anti-lipopolysaccharide factors (ALFs), a type of cationic antimicrobial peptides (AMPs), and their derivatives are becoming predominant candidates for potential drugs in viral and bacterial diseases. This study reports the first ALF from the mud crab Scylla tranquebarica (StALF, JQ899453) and the second ALF isoform from the blue swimmer crab Portunus pelagicus (PpALF2, JQ899452). Both sequences encoded for precursor molecules, starting with a signal peptide containing 26 amino acid residues, followed by a highly cationic mature peptide, containing two conserved cysteine residues flanking a putative lipopolysaccharide (LPS)-binding domain. BLAST analysis revealed that both PpALF2 and StALF exhibited significant similarity with crustacean ALF sequences. The predicted molecular mass of the mature ALFs was 11.2 kDa with an estimated pI of 10.0. PpALF2 and StALF also showed the typical pattern of alternating hydrophobic and hydrophilic residues in their putative disulphide loop, suggesting that they comprise the same functional domain. Phylogenetic analysis showed that PpALF2 and StALF have similar evolutionary status and they were phylogenetically ancient immune effector molecules which may play an essential role in the host defense mechanism. The spatial structures of PpALF2 and StALF possessed four beta-strands and two alpha-helices. The results indicated that there were more than one ALF involved in crab immunity against various pathogens. ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control in crustacean aquaculture

Follicle-stimulating hormone isoforms and plasma concentrations of estradiol and inhibin A in dairy cows with ovulatory and non-ovulatory follicles during the first postpartum follicle wave

Following parturition, all cows display a wave of ovarian follicular growth, but a large proportion fail to generate a preovulatory rise in estradiol, and hence fail to ovulate. Follicle-stimulating hormone (FSH) exists as multiple isoforms in the circulation depending on the type and extent of glycosylation, and this has pronounced effects on its biological properties. This study examined differences in plasma FSH, estradiol, and inhibin A concentrations, and the distribution of FSH isoforms in cows with ovulatory or atretic dominant follicles during the first postpartum follicle wave. Plasma FSH isoform distribution was examined in both groups during the period of final development of the dominant follicle by liquid phase isoelectric focusing. Cows with an ovulatory follicle had higher circulating estradiol and inhibin A concentrations, and lower plasma FSH concentrations. The distribution of FSH isoforms displayed a marked shift toward the less acidic isoforms in cows with ovulatory follicles. A higher proportion of the FSH isoforms had a pl>5.0 in cows with ovulatory follicles compared to those with atretic follicles. In addition, cows with ovulatory follicles had greater dry matter intake, superior energy balance, elevated circulating concentrations of insulin and insulin-like growth factor-I, and lower plasma nonesterified fatty acids. The shift in FSH isoforms toward a greater abundance of the less acidic isoforms appears to be a key component in determining the capability for producing a preovulatory rise in estradiol, and this shift in FSH isoforms was associated with more favorable bioenergetic and metabolic status. (C) 2008 Elsevier Inc. All rights reserved.