Reciprocal amplification of caspase-3 activity by nuclear export of a putative human RNA-modifying protein, PUS10 during TRAIL-induced apoptosis.

Pus10 is a pseudouridine synthase present in Archaea and Eukarya, but not in Bacteria and yeast. It has been suggested that the human PUS10 (DOBI) gene is needed during TRAIL-induced apoptosis. We analyzed the role of PUS10 in TRAIL-induced apoptosis by immunofluorescence, immunoblotting and several indicators of apoptosis. We examined several TRAIL-sensitive cell lines and we also examined some resistant cell lines after treatment with cycloheximide. PUS10 is mainly present in the nucleus. Early during apoptosis, PUS10 translocates to mitochondria via CRM1-mediated export with the concurrent release of cytochrome c and SMAC. Caspase-3 is required for PUS10 translocation, which reciprocally amplifies the activity of caspase-3 through the intrinsic/mitochondrial pathway. This suggests that in addition to cytoplasmic factors, nuclear factors also have a direct role in the major apoptosis pathways. However, p53 is not involved in TRAIL-induced PUS10 movement. The caspase-3-mediated movement of PUS10 and the release of mitochondrial contents enhancing caspase-3 activity creates a feedback amplification loop for caspase-3 action. Therefore, any defect in the movement or interactions of PUS10 would reduce the TRAIL sensitivity of tumor cells

Role of forefinger and thumb loops in production of Ψ54 and Ψ55 in tRNAs by archaeal Pus10

Pseudouridines (Ψ) are found in structurally and functionally important regions of RNAs. Six families of Ψ synthases, TruA, TruB, TruD, RsuA, RluA, and Pus10 have been identified. Pus10 is present in Archaea and Eukarya. While most archaeal Pus10 produce both tRNA Ψ54 and Ψ55, some produce only Ψ55. Interestingly, human PUS10 has been implicated in apoptosis and Crohn\u27s and Celiac diseases. Homology models of archaeal Pus10 proteins based on the crystal structure of human PUS10 reveal that there are subtle structural differences in all of these Pus10 proteins. These observations suggest that structural changes in homologous proteins may lead to loss, gain, or change of their functions, warranting the need to study the structure-function relationship of these proteins. Using comparison of structural models and a series of mutations, we identified forefinger loop (reminiscent of that of RluA) and an Arg and a Tyr residue of archaeal Pus10 as critical determinants for its Ψ54, but not for its Ψ55 activity. We also found that a Leu residue, in addition to the catalytic Asp, is essential for both activities. Since forefinger loop is needed for both rRNA and tRNA Ψ synthase activities of RluA, but only for tRNA Ψ54 activity of Pus10, archaeal Pus10 proteins must use a different mechanism of recognition for Ψ55 activity. We propose that archaeal Pus10 uses two distinct mechanisms for substrate uridine recognition and binding. However, since we did not observe any mutation that affected only Ψ55 activity, both mechanisms for archaeal Pus10 activities must share some common features

Role of forefinger and thumb loops in production of 54 and 55 in tRNAs by archaeal Pus10

Joardar A, Jana S, Fitzek E, et al. Role of forefinger and thumb loops in production of  54 and  55 in tRNAs by archaeal Pus10. RNA. 2013;19(9):1279-1294

Psychosocial impact of undergoing prostate cancer screening for men with BRCA1 or BRCA2 mutations.

OBJECTIVES: To report the baseline results of a longitudinal psychosocial study that forms part of the IMPACT study, a multi-national investigation of targeted prostate cancer (PCa) screening among men with a known pathogenic germline mutation in the BRCA1 or BRCA2 genes. PARTICPANTS AND METHODS: Men enrolled in the IMPACT study were invited to complete a questionnaire at collaborating sites prior to each annual screening visit. The questionnaire included sociodemographic characteristics and the following measures: the Hospital Anxiety and Depression Scale (HADS), Impact of Event Scale (IES), 36-item short-form health survey (SF-36), Memorial Anxiety Scale for Prostate Cancer, Cancer Worry Scale-Revised, risk perception and knowledge. The results of the baseline questionnaire are presented. RESULTS: A total of 432 men completed questionnaires: 98 and 160 had mutations in BRCA1 and BRCA2 genes, respectively, and 174 were controls (familial mutation negative). Participants' perception of PCa risk was influenced by genetic status. Knowledge levels were high and unrelated to genetic status. Mean scores for the HADS and SF-36 were within reported general population norms and mean IES scores were within normal range. IES mean intrusion and avoidance scores were significantly higher in BRCA1/BRCA2 carriers than in controls and were higher in men with increased PCa risk perception. At the multivariate level, risk perception contributed more significantly to variance in IES scores than genetic status. CONCLUSION: This is the first study to report the psychosocial profile of men with BRCA1/BRCA2 mutations undergoing PCa screening. No clinically concerning levels of general or cancer-specific distress or poor quality of life were detected in the cohort as a whole. A small subset of participants reported higher levels of distress, suggesting the need for healthcare professionals offering PCa screening to identify these risk factors and offer additional information and support to men seeking PCa screening

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

STRUCTURAL AND FUNCTIONAL STUDIES OF ARCHAEAL BOX C/D GUIDE RNA AND ROLE OF A PUTATIVE HUMAN PSEUDOURIDINE SYNTHASE, PUS10 IN APOPTOSIS

RNAs undergo different posttranscriptional chemical modifications, which affect their structural stability and functional diversity. RNA methylation is a very common type of post-transcriptional modification and is present in all domains of life: Archaea, Eukaryotes and Bacteria. Some of these methylations are catalyzed either by a RNA-protein complex or by stand-alone enzymes. The RNA-protein complex (Ribonucleoprotein complex) is comprised of a small RNA known as the guide RNA (Box C/D RNA) and core proteins (L7Ae, Nop5, and Fibrillarin). Box C/D RNAs contain conserved regions, called box C and box D near their 5’ and 3’ termini, respectively, and their imperfect copies called box C’ and box D’, internally. A short stretch of sequence between these Boxes are known as the guide/spacer regions, as the guide region helps in recruiting and positioning a specific target RNA for modification. Both in Archaea and Eukarya, box C and box D, as well as box C’ and box D’ together can form a structure called a Kink-turn (K-turn) that is characterized by a canonical Watson-Crick base-paired stem on one side, and a non-canonical stem on the other, separated by a 3-nucleotide loop. In Archaea box C’ and D’ can also form a K-loop, where the canonical stem of K-turn is replaced by a loop. Archaeal L7Ae binds first to the K-turn or K-loop and allows the recruitment of other proteins to form the complex. The presence of a unique box C/D RNA of Haloferax volcanii, called sR-tMet has been reported previously to guide the 2’-O-methylation of C34 in elongator pre-tRNAMet. Here we tried to characterize the structure-function relationship of this guide RNA under in vivo conditions. This RNA lacks a conventional K-turn or K-loop at its C’/D’ motif. We have created an H. volcanii strain that has a genomic deletion of sR-tMet. The sR-tMet gene is not essential for H. volcanii but this sR-tMet deleted strain lacks the 2’-O-methylation of C34 of its elongator tRNAMet. Unlike the close sR-tMet homologs (sR8 from Methanocaldococcus jannaschii and sR49 from Pyrococcus abyssi), the Box C’/D’ motif of sR-tMet is neither a K-turn nor a K-loop. The introduction of proper K-loop in the Box C’/D’ motif (sR-tMet with K-loop) abolished its Cm34 modification function in ΔsR-tMet strain. Direct interaction between L7Ae and the K-loop is not an absolute requirement for its function. However, disruption of the G/A and A/G pairing in Box C/D motif and Box D’ suggests the importance of these non-Watson crick base pairings in respect to sR-tMet’s function. Several other mutational studies have revealed that peculiar sR-tMet guide RNA from H. volcanii, behaves more like a Eukaryotic Box C/D RNA (where the K-loop is not required and presence of longer spacer length) than regular Archaeal one. Pseudouridine synthase 10 (Pus10) is the most recently identified Ψ synthase, found only in higher eukaryotes and Archaea. Archaeal Pus10 produces either tRNA Ψ55 or both tRNA Ψ54 and Ψ55 modifications. In Human, its Ψ synthase activity is not yet confirmed and interestingly it has been implicated in apoptosis. Herein for the first time we revealed that this putative RNA Ψ synthase protein, Human Pus10 (HuP10), translocates from the nucleus to the cytoplasm in TRAIL induced apoptosis. This nucleo-cytoplasmic movement of HuP10 occurs through the CRM1 mediated nuclear export pathway and Caspase 3 influences this movement. HuP10 also mediates crosstalk between the extrinsic and intrinsic pathways during TRAIL-induced apoptosis. Other than its involvement in apoptosis, we have also uncovered that HuP10 is involved in regulation of cell proliferation. Depletion (knockdown) of this protein in different cancer cell lines, promotes cell migration and anchorage-independent cell growth in the absence of any apoptotic stimulation

Procathepsin D and cancer: From molecular biology to clinical applications

Procathepsin D (pCD) is overexpressed and secreted by cells of various tumor types including breast and lung carcinomas. pCD affects multiple features of tumor cells including proliferation, invasion, metastases and apoptosis. Several laboratories have previously shown that the mitogenic effect of pCD on cancer cells is mediated via its propeptide part (APpCD). However, the exact mechanism of how pCD affects cancer cells has not been identified. Recent observations have also revealed the possible use of pCD/APpcD as a marker of cancer progression. The purpose of this review is to summarize the three major potentials of pCD-tumor marker, potential drug, and screening agent

Rhodamine-azobenzene based single molecular probe for multiple ions sensing: Cu 2+ , Al 3+ , Cr 3+ and its imaging in human lymphocyte cells

A photoinduced electron transfer (PET) and chelation-enhanced fluorescence (CHEF) regulated rhodamineazobenzene chemosensor (L) was synthesized for chemoselective detection of Al3+, Cr3+, and Cu2+ by UV-Visible absorption study whereas Al3+ and Cr3+ by Iluorimetric study in EtOH-H2O solvent. L showed a clear fluorescence emission enhancement of 21 and 16 fold upon addition of Al3+ and Cr3+ due to the 1:1 host-guest complexation, respectively. This is first report on rhodamine-azobenzene based Cr3+ chemosensor. The complex formation, restricted imine isomerization, inhibition of PET (photo-induced electron transfer) process with the concomitant opening of the spirolactam ring induced a turn-on fluorescence response. The higher binding constants 6.7 x 10(3) M-1 and 3.8 x 10(3) M-1 for Al(3+ )and Cr3+, respectively and lower detection limits 1 x 10(-6)M and 2 x 10(-6) M for Al3+ and Cr3+, respectively in a buffered solution with high reversible nature describes the potential of L as an effective tool for detecting Al3+ and Cr3+ in a biological system with higher intracellular resolution. Finally, L was used to map the intracellular concentration of Al3+ and Cr3+ in human lymphocyte cells (HLCs) at physiological pH very effectively. Altogether, our findings will pave the way for designing new chemosensors for multiple analytes and those chemosensors will be effective for cell imaging study