Anirban Ghosh

Regulation of Human p53 Activity and Cell Localization by Alternative Splicing

ABSTRACT The development of cancer is a multistep process involving mutations in proto-oncogenes, tumor suppressor genes, and other genes which control cell proliferation, telomere stability, angiogenesis, and other complex traits. Despite this complexity, the cellular pathways controlled by the p53 tumor suppressor protein are compromised in most, if not all, cancers. In normal cells, p53 controls cell proliferation, senescence, and/or mediates apoptosis in response to stress, cell damage, or ectopic oncogene expression, properties which make p53 the prototype tumor suppressor gene. Defining the mechanisms of regulation of p53 activity in normal and tumor cells has therefore been a major priority in cell biology and cancer research. The present study reveals a novel and potent mechanism of p53 regulation originating through alternative splicing of the human p53 gene resulting in the expression of a novel p53 mRNA. This novel p53 mRNA encodes an N-terminally deleted isoform of p53 termed p47. As demonstrated within, p47 was able to effectively suppress p53-mediated transcriptional activity and impair p53-mediated growth suppression. It was possible to select for p53-null cells expressing p47 alone or coexpressing p53 in the presence of p47 but not cells expressing p53 alone. This showed that p47 itself does not suppress cell viability but could control p53-mediated growth suppression. Interestingly, p47 was monoubiquitinated in an Mdm2-independent manner, and this was associated with its export out of the nucleus. In the presence of p47, there was a reduction in Mdm2-mediated polyubiquitination and degradation of p53, and this was also associated with increased monoubiquitination and nuclear export of p53. The expression of p47 through alternative splicing of the p53 gene thus has a major influence over p53 activity at least in part through controlling p53 ubiquitination and cell localization.

Immunization with A2 protein results in a mixed Th1/Th2 and a humoral response which protects mice against Leishmania donovani infections

The A2 genes of Leishmania donovani encode amastigote-specific A2 proteins, which are considered to be virulence factors required for the survival of this protozoan parasite in the mammalian host. The A2 genes are present within a multigene family and corresponding A2 proteins are composed predominantly of multiple copies of a 10 amino acid repeat sequences. A2-specific antibodies have been detected in the sera of patients suffering from visceral leishmaniasis (VL) and it has been shown that generation of A2 deficient L. donovani resulted in an avirulent phenotype. In this report, we show that immunization of mice with recombinant A2 protein conferred significant protection against challenge infection with L. donovani. The protection correlated with in vitro splenocyte proliferation, production of IFN-gamma in response to A2 protein and the presence of A2-specific antibodies in the sera of immunized mice. These data demonstrate that A2 represents a potential antigen for protection against infection with L. donovani and VL.

Protection against Leishmania donovani infection by DNA vaccination : increased DNA vaccination efficiency through inhibiting the cellular p53 response

DNA-vaccination holds great promise for the future of vaccine development against infectious diseases, especially in developing countries. We therefore investigated the possibility of using DNA-vaccination against Leishmania donovani infection with the A2 virulence gene and whether inhibiting the cellular p53 response could increase the effectiveness of the A2 DNA vaccine. p53, also known as the guardian of the genome, is activated following DNA transfection and has pleotropic effects on cells, which could have adverse effects on the effectiveness of DNA-vaccination. Two major observations are reported within. First, vaccination with the A2 gene induced both humoral and cellular immune responses against A2 which provided significant protection against infection with L. donovani. Second, inhibition of p53 with human papillomavirus E6 resulted in higher expression of heterologous transfected genes in vitro and more efficient DNA-vaccination in vivo. These results have important implications for DNA vaccination against leishmaniasis and potentially against other infectious diseases.

Immunization with recombinant LD1 antigens protects against experimental leishmaniasis

The genes, ORFF and BT1 (previously ORFG), are part of the multigenic LD1 locus on chromosome 35 which is frequently amplified in Leishmania. BT1 encodes a biopterin transporter, while the function of the ORFF gene product is unknown, but it is localized to the nucleus. We show here that immunization of mice with recombinant ORFF and BT1 proteins, individually, or in combination, conferred partial protection against challenge with Leishmania donovani. Protection correlated with the production of antigen-specific antibodies and in vitro splenocyte proliferation. Thus, these antigens can be potential vaccine candidates against visceral leishmaniasis.

Involvement of Nuclear Export in Human Papillomavirus Type 18 E6-Mediated Ubiquitination and Degradation of p53

ABSTRACT The E6 protein from high-risk human papillomaviruses (HPVs) targets the p53 tumor suppressor for degradation by the proteasome pathway. This ability contributes to the oncogenic potential of these viruses. However, several aspects concerning the mechanism of E6-mediated p53 degradation at the cellular level remain to be clarified. This study therefore examined the role of cell localization and ubiquitination in the E6-mediated degradation of p53. As demonstrated within, following coexpression both p53 and high-risk HPV type 18 (HPV-18) E6 (18E6) shuttle from the nucleus to the cytoplasm. Mutation of the C-terminal nuclear export signal (NES) of p53 or treatment with leptomycin B inhibited the 18E6-mediated nuclear export of p53. Impairment of nuclear export resulted in only a partial reduction in 18E6-mediated degradation, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation. This was also consistent with the observation that 18E6 mediated the accumulation of polyubiquitinated p53 in the nucleus. In comparison, a p53 isoform that localizes predominantly to the cytoplasm was not targeted for degradation by 18E6 in vivo but could be degraded in vitro, arguing that nuclear p53 is the target for E6-mediated degradation. This study supports a model in which (i) E6 mediates the accumulation of polyubiquitinated p53 in the nucleus, (ii) E6 is coexported with p53 from the nucleus to the cytoplasm via a CRM1 nuclear export mechanism involving the C-terminal NES of p53, and (iii) E6-mediated p53 degradation can be mediated by both nuclear and cytoplasmic proteasomes.

Identification and Characterization of a Protein-tyrosine Phosphatase in Leishmania INVOLVEMENT IN VIRULENCE

Leishmania parasites are eukaryotic protozoans responsible for a variety of human diseases known as leishmaniasis, which ranges from skin lesions to fatal visceral infections. Leishmania is transmitted by the bite of an infected sandfly where it exists as promastigotes and, upon entry into a mammalian host, differentiates into amastigotes, which replicate exclusively in macro-phages. The biochemical pathways enabling Leishmania to differentiate and survive in the mammalian host are poorly defined. We have therefore examined the role of protein-tyrosine phosphorylation, which is essential in regulating cell function in higher eukaryotes. Using the recently completed Leishmania genome, we have identified and cloned a Leishmania protein-tyrosine phosphatase (PTP) gene (LPTP1) by virtue of its homology with the human protein-tyrosine phosphatase 1B gene (hPTP1B). The enzyme activity of recombinant LPTP1 was confirmed using a combination of PTP-specific substrates and inhibitors. We further demonstrate, by creating LPTP1 null mutants through gene targeting, that LPTP1 is necessary for survival as amastigotes in mice, but it is dispensable for survival as promastigotes in culture. Human PTPs, including the PTP1B enzyme, are actively pursued drug targets for a variety of diseases. The observations with the LPTP1 mutants in mice suggest that it may also represent a drug target against the mammalian amastigote stage. However, in silico structure analysis of LPTP1 revealed a striking similarity with hPTP1B in the active site suggesting that, although this is an attractive drug target, it may be difficult to develop an inhibitor specific for the Leishmania LPTP1.

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein A POTENTIAL MECHANISM PROMOTING HUMAN CANCER

Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.

Heterologous expression of a mammalian protein tyrosine phosphatase gene in Leishmania: effect on differentiation

Leishmania is a protozoan pathogen which is transmitted to humans through the bite of an infected sandfly. This infection results in a spectrum of diseases throughout the developing world, collectively known as leishmaniasis. During its life cycle, Leishmania differentiates from the promastigote stage in the sandfly vector into the amastigote stage in the mammalian host where it multiplies exclusively in macrophage phagolysosomes. Although differentiation of Leishmania is essential for its survival and pathogenesis in the mammalian host, this process is poorly understood. In higher eukaryotic cells, protein tyrosine phosphorylation plays a central role in cell proliferation, differentiation and overall function. We have therefore investigated the role of protein tyrosine phosphorylation in Leishmania differentiation by undertaking complementary approaches to mediate protein tyrosine dephosphorylation in vivo. In the present study, L. donovani were engineered to express a mammalian protein tyrosine phosphatase, or were treated with inhibitors of protein tyrosine kinases, and the resulting phenotype was examined. Both approaches resulted in a partial differentiation from promastigotes to amastigotes including the expression of the amastigote specific A2 protein, morphological change and increased virulence. These data provide support for the involvement of tyrosine phosphorylation in the differentiation of Leishmania.