83 research outputs found
Translational regulation of human methionine synthase by upstream open reading frames
No full textOltean, Sebastian/0000-0001-7890-8439WOS: 000250235600002PubMed ID: 17683808Methionine synthase is a key enzyme poised at the intersection of folate and sulfur metabolism and functions to reclaim homocysteine to the methionine cycle. The 5' leader sequence in human MS is 394 nucleotides long and harbors two open reading frames (uORFs). In this study, regulation of the main open reading frame by the uORFs has been elucidated. Both uORFs downregulate translation as demonstrated by mutation of the upstream AUG codons (uAUG) either singly or simultaneously. The uAUGs are capable of recruiting the 40S ribosomal complex as revealed by their ability to drive reporter expression in constructs in which the luciferase is fused to the uORFs. uORF2, which is predicted to encode a 30 amino acid long polypeptide, has a clustering of rare codons encoding arginine and proline. Mutation of a tandemly repeated rare codon for arginine at positions 3 and 4 in uORF2 to either common codons for the same amino acid or common codons for alanine results in complete alleviation of translation inhibition. This suggests a mechanism for ribosome stalling and demonstrates that the cis-effects on translation by uORF2 is dependent on the nucleotide sequence but is apparently independent of the sequence of the encoded peptide. This study reveals complex regulation of the essential housekeeping gene, methionine synthase, by the uORFs in its leader sequence. (c) 2007 Elsevier B.V. All rights reserved.NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASESUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) [R01DK045776, R01DK064959, R01DK045776, R01DK064959, R01DK045776, R01DK064959, R01DK045776, R01DK045776, R01DK064959, R01DK045776, R01DK045776, R01DK064959, R01DK045776, R01DK045776, R01DK045776, R01DK045776, R01DK045776, R01DK045776, R01DK064959, R01DK045776, R01DK045776, R01DK064959, R01DK045776, R01DK045776, R01DK045776, R01DK045776, R01DK045776, R01DK045776, R01DK045776, R01DK045776, R01DK045776, R01DK045776] Funding Source: NIH RePORTER; NIDDK NIH HHSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) [R01 DK045776-18, R01 DK064959, DK64959, R01 DK045776] Funding Source: Medlin
Modulation of VEGF-A alternative splicing as a novel treatment in chronic kidney disease.
No full textThis is the final version of the article. Available from MDPI via the DOI in this record.Vascular endothelial growth factor A (VEGF-A) is a prominent pro-angiogenic and pro-permeability factor in the kidney. Alternative splicing of the terminal exon of VEGF-A through the use of an alternative 3' splice site gives rise to a functionally different family of isoforms, termed VEGF-Axxxb, known to have anti-angiogenic and anti-permeability properties. Dysregulation of the VEGF-Axxx/VEGF-Axxxb isoform balance has recently been reported in several kidney pathologies, including diabetic nephropathy (DN) and Denys-Drash syndrome. Using mouse models of kidney disease where the VEGF-A isoform balance is disrupted, several reports have shown that VEGF-A165b treatment/over-expression in the kidney is therapeutically beneficial. Furthermore, inhibition of certain splice factor kinases involved in the regulation of VEGF-A terminal exon splicing has provided some mechanistic insight into how VEGF-A splicing could be regulated in the kidney. This review highlights the importance of further investigation into the novel area of VEGF-A splicing in chronic kidney disease pathogenesis and how future studies may allow for the development of splicing-modifying therapeutic drugs.This work was supported by grants to Seb Oltean from British Heart Foundation (PG/15/53/31371), Richard Bright VEGF Research Trust and Diabetes UK (17/000568). These grants include funds for Open Access publishing
Gene-nutrient interactions in homocysteine metabolism: Regulation of human methionine synthase by B12
No full textStudies spanning the last decade have linked elevated plasma homocysteine with increased risk for cardiovascular and other diseases. Homocysteine is a sulfur containing amino acid and its metabolism is directly controlled by the activity of three enzymes: B6 dependent cystathionine beta-synthase, B12-dependent methionine synthase (MS), and betaine homocysteine methyl transferase. Homocysteine can either condense with serine in a reaction catalyzed by cystathionine beta-synthase or be methylated to methionine in a reaction catalyzed by betaine homocysteine methyl transferase (with limited tissue distribution) or by MS. Several studies indicate the benefit of multivitamin (B6, B12 and folate) treatment in lowering plasma homocysteine. Mutations in MS are correlated with hereditary hyperhomocysteinemia. It was first reported over 30 years ago that the activity of MS in cultured cells was enhanced several fold upon supplementation with vitamin B12 . However, the mechanism of this activation was not elucidated. It is important to emphasize that the B12 effect was not incurred by a change in the culture conditions from B12 depleted to a B 12 replete medium but rather by B12 supplementation of medium already containing “normal” B12 levels. Previous work done in our laboratory revealed that this regulation occurs at a post-transcriptional level. In my studies I have demonstrated that B12 supplementation does not affect either MS mRNA stability or MS protein turnover, but increases translation of methionine synthase by unmasking mRNA molecules from the ribonucleoprotein pool and shifting them to the polysomal pool. Reporter gene constructs have revealed that an element in the 5′ untranslated region (UTR) of the MS mRNA is involved in mediating the B12 response. Furthermore, I have demonstrated that the MS 5′UTR contains an internal ribosome entry site, that is modulated by B12. Structure probing analysis of the 5′UTR has proved that B12 does not bind directly to the mRNA. Instead it probably exerts its action with the help of a protein. The observed increase in the metabolic flux through methionine synthase in the presence of B12 suggests a mechanism for lowering of homocysteine levels by B12
Modulators of alternative splicing as novel therapeutics in cancer
No full textAlternative splicing (AS), the process of removing introns from pre-mRNA and re-arrangement of exons to give several types of mature transcripts, has been described more than 40 years ago. However, until recently, it has not been clear how extensive it is. Genome-wide studies have now conclusively shown that more than 90% of genes are alternatively spliced in humans. This makes AS one of the main drivers of proteomic diversity and, consequently, determinant of cellular function repertoire. Unsurprisingly, given its extent, numerous splice isoforms have been described to be associated with several diseases including cancer. Many of them have antagonistic functions, e.g., pro- and anti-angiogenic or pro- and anti-apoptotic. Additionally several splice factors have been recently described to have oncogene or tumour suppressors activities, like SF3B1 which is frequently mutated in myelodysplastic syndromes. Beside the implications for cancer pathogenesis, de-regulated AS is recognized as one of the novel areas of cell biology where therapeutic manipulations may be designed. This editorial discusses the possibilities of manipulation of AS for therapeutic benefit in cancer. Approaches involving the use of oligonucleotides as well as small molecule splicing modulators are presented as well as thoughts on how specificity might be accomplished in splicing therapeutics.</p
Targeting Angiogenesis in Prostate Cancer
No full textThis is the author accepted manuscript. The final version is available from MDPI via the DOI in this record.Prostate cancer is the most commonly diagnosed cancer among men in the Western world. Although localized disease can be effectively treated with established surgical and radiopharmaceutical treatments options, the prognosis of castration-resistant advanced prostate cancer is still disappointing. The objective of this study was to review the role of angiogenesis in prostate cancer and to investigate the effectiveness of anti-angiogenic therapies. A literature search of clinical trials testing the efficacy of anti-angiogenic therapy in prostate cancer was performed using Pubmed. Surrogate markers of angiogenic activity (microvessel density and vascular endothelial growth factor A (VEGF-A) expression) were found to be associated with tumor grade, metastasis, and prognosis. Six randomizedstudies were included in this review: two phase II trials on localized and hormone-sensitive disease (n = 60 and 99 patients) and four phase III trials on castration-resistant refractory disease (n = 873 to 1224 patients). Although the phase II trials showed improved relapse-free survival and stabilisation of the disease, the phase III trials found increased toxicity and no significant improvement in overall survival. Although angiogenesis appears to have an important role in prostate cancer, the results of anti-angiogenic therapy in castration-resistant refractory disease have hitherto been disappointing. There are various possible explanations for this lack of efficacy in castration-resistant refractory disease: redundancy of angiogenic pathways, molecular heterogeneity of the disease, loss of tumor suppressor protein phosphatase and tensin homolog (PTEN) expression as well as various VEGF-A splicing isoforms with pro- and anti-angiogenic activity. A better understanding of the molecular mechanisms of angiogenesis may help to develop effective anti-angiogenic therapy in prostate cancer.British Heart FoundationDiabetes U
Alternative Splicing in CKD
No full textAlternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with ≥94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion of splice isoforms (or novel isoforms), without an apparent driver mutation. It is therefore essential to study how AS is regulated in physiology, how it contributes to pathogenesis, and whether we can manipulate faulty splicing for therapeutic advantage. Although the disease most commonly linked to deregulation of AS in several genes is cancer, many reports detail pathogenic splice variants in diseases ranging from neuromuscular disorders to diabetes or cardiomyopathies. A plethora of splice variants have been implicated in CKDs as well. In this review, we describe examples of these CKD-associated splice variants and ideas on how to manipulate them for therapeutic benefit.</p
The many faces of SRPK1
No full textSerine-arginine protein kinase 1 (SRPK1) phosphorylates proteins involved in the regulation of several mRNA processing pathways including alternative splicing. SRPK1 has been recently reported to be over-expressed in multiple cancers including prostate, breast, lung and glioma. Several studies have shown that inhibition of SRPK1 has anti-tumoural effects and consequently SRPK1 has become a new candidate for targeted therapies. Interestingly, in terms of molecular mechanism, SRPK1 seems to act heterogeneously and has been reported to affect several processes in different cancers, for example angiogenesis in prostate and colon cancer, apoptosis in breast and colon cancer and migration in breast cancer. A recent report adds to this puzzle, showing that the main effect of overexpression of SRPK1 in non-small-cell lung carcinoma is to stimulate a stem cell-like phenotype. This pleiotropy might be related to preferential activation of different downstream signaling pathways by SRPK1 in various cancers
SRPK1 inhibition in prostate cancer:a novel anti-angiogenic treatment through modulation of VEGF alternative splicing
No full textProstate cancer remains one of the leading causes of cancer death in men around the world, regardless of intense research and development of novel therapies in the last 10 years. One of the new avenues that has been tested − inhibition of angiogenesis − has been disappointing so far in clinical studies in spite of strong evidence that determinants of angiogenesis (e.g vascular endothelial growth factor) are strongly associated with disease progression. One of the reasons for these outcomes may be our poor understanding of the biology of angiogenesis in prostate cancer (and probably other cancers as well) resulting in inhibition of both detrimental and favourable molecules. We discuss here novel targeted and more specific approaches to inhibit angiogenesis in prostate cancer as well as a completely new therapeutic modality to do this − modulation of alternative splicing − that may be applicable to other molecules/biological processes as well
Aberrant Splicing as a Mechanism for Resistance to Cancer Therapies
No full textCancer is biologically diverse, highly heterogeneous, and associated with molecular alterations, significantly contributing to mortality worldwide. Currently, cancer patients are subjected to single or combination treatments comprising chemotherapy, surgery, immunotherapy, radiation therapy, and targeted therapy. Chemotherapy remains the first line of treatment in cancer but faces a major obstacle in the form of chemoresistance. This obstacle has resulted in relapses and poor patient survival due to decreased treatment efficacy. Aberrant pre-mRNA alternative splicing can significantly modulate gene expression and function involved in the resistance mechanisms, potentially shaping the intricate landscape of tumour chemoresistance. Thus, novel strategies targeting abnormal pre-mRNA alternative splicing and understanding the molecular mechanisms of chemotherapy resistance could aid in overcoming the chemotherapeutic challenges. This review first highlights drug targets, drug pumps, detoxification mechanisms, DNA damage response, and evasion of apoptosis and cell death as key molecular mechanisms involved in chemotherapy resistance. Furthermore, the review discusses the progress of research on the dysregulation of alternative splicing and molecular targets involved in chemotherapy resistance in major cancer types
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