Isolation and characterization of Pas2p, a peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris

The pas2 mutant of the methylotrophic yeast Pichia pastoris is characterized by a deficiency in peroxisome biogenesis. We have cloned the PpPAS2 gene by functional complementation and show that it encodes a protein of 455 amino acids with a molecular mass of 52 kDa. In a Pppas2 null mutant, import of both peroxisomal targeting signal 1 (PTS1)- and PTS2-containing proteins is impaired as shown by biochemical fractionation and fluorescence microscopy. No morphologically distinguishable peroxisomal structures could be detected by electron microscopy in Pppas2 null cells induced on methanol and oleate, suggesting that PpPas2p is involved in the early stages of peroxisome biogenesis. PpPas2p is a peroxisomal membrane protein (PMP) and is resistant to extraction by 1 M NaCl or alkaline sodium carbonate, suggesting that it is a peroxisomal integral membrane protein. Two hydrophobic domains can be distinguished which may be involved in anchoring PpPas2p to the peroxisomal membrane. PpPas2p is homologous to the Saccharomyces cerevisiae Pas3p. The first 40 amino acids of PpPas2p, devoid of the hydrophobic domains, are sufficient to target a soluble fluorescent reporter protein to the peroxisomal membrane, with which it associates tightly, A comparison with the membrane peroxisomal targeting signal of PMP47 of Candida boidinii revealed a stretch of positively charged amino acids common to both sequences. The role of peroxisomal membrane targeting signals and transmembrane domains in anchoring PMPs to the peroxisomal membrane is discussed.</p

Recruitment of the Major Vault Protein by InlK: A Listeria monocytogenes Strategy to Avoid Autophagy

L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK− bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles

Non-coding rnas, a novel paradigm for the management of gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal malignancies found in the gastrointestinal tract. At a molecular level, most GISTs are characterized by gain-of-function mutations in V-Kit Hardy–Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog (KIT) and Platelet Derived Growth Factor Receptor Alpha (PDGFRA), leading to constitutive activated signaling through these receptor tyrosine kinases, which drive GIST pathogenesis. In addition to surgery, treatment with the tyrosine kinase inhibitor imatinib forms the mainstay of GIST treatment, particularly in the advanced setting. Nevertheless, the majority of GISTs develop imatinib resistance. Biomarkers that indicate metastasis, drug resistance and disease progression early on could be of great clinical value. Likewise, novel treatment strategies that overcome resistance mechanisms are equally needed. Non-coding RNAs, particularly microRNAs, can be employed as diagnostic, prognostic or predictive biomarkers and have therapeutic potential. Here we review which non-coding RNAs are deregulated in GISTs, whether they can be linked to specific clinicopathological features and discuss how they can be used to improve the clinical management of GISTs

Role of Lipid Rafts in Pathogen-Host Interaction: A Mini Review

Lipid rafts, also known as microdomains, are important components of cell membranes and are enriched in cholesterol, glycophospholipids and receptors. They are involved in various essential cellular processes, including endocytosis, exocytosis and cellular signaling. Receptors are concentrated at lipid rafts, through which cellular signaling can be transmitted. Pathogens exploit these signaling mechanisms to enter cells, proliferate and egress. However, lipid rafts also play an important role in initiating antimicrobial responses by sensing pathogens via clustered pathogen-sensing receptors and triggering downstream signaling events such as programmed cell death or cytokine production for pathogen clearance. In this review, we discuss how both host and pathogens use lipid rafts and associated proteins in an arms race to survive. Special attention is given to the involvement of the major vault protein, the main constituent of a ribonucleoprotein complex, which is enriched in lipid rafts upon infection with vaccinia virus

Tissue-Specific Chk1 Activation Determines Apoptosis by Regulating the Balance of p53 and p21

Summary: The DNA damage response (DDR) protects cells against genomic instability. Surprisingly, little is known about the differences in DDR across tissues, which may affect cancer evolutionary trajectories and chemotherapy response. Using mathematical modeling and quantitative experiments, we found that the DDR is regulated differently in human breast and lung primary cells. Equal levels of cisplatin-DNA lesions caused stronger Chk1 activation in lung cells, leading to resistance. In contrast, breast cells were more resistant and showed more Chk2 activation in response to doxorubicin. Further analyses indicate that Chk1 activity played a regulatory role in p53 phosphorylation, whereas Chk2 activity was essential for p53 activation and p21 expression. We propose a novel “friction model,” in which the balance of p53 and p21 levels contributes to the apoptotic response in different tissues. Our results suggest that modulating the balance of p53 and p21 dynamics could optimize the response to chemotherapy. : Bioinformatics; Mathematical Biosciences; Systems Biology; Cancer Systems Biology Subject Areas: Bioinformatics, Mathematical Biosciences, Systems Biology, Cancer Systems Biolog

Major Vault Protein, a Candidate Gene in 16p11.2 Microdeletion Syndrome, Is Required for the Homeostatic Regulation of Visual Cortical Plasticity

Microdeletion of a region in chromosome 16p11.2 increases susceptibility to autism. Although this region contains exons of 29 genes, disrupting only a small segment of the region, which spans five genes, is sufficient to cause autistic traits. One candidate gene in this critical segment is MVP, which encodes for the major vault protein (MVP) that has been implicated in regulation of cellular transport mechanisms. MVP expression levels in MVP+/- mice closely phenocopy those of 16p11.2 mutant mice, suggesting that MVP+/- mice may serve as a model of MVP function in 16p11.2 microdeletion. Here we show that MV Pregulates the homeostatic component of ocular dominance (OD) plasticity in primary visual cortex. MVP+/- mice of both sexes show impairment in strengthening of open-eye responses after several days of monocular deprivation (MD), whereas closed-eye responses are weakened as normal, resulting in reduced overall OD plasticity. The frequency of miniature EPSCs (mEPSCs) in pyramidal neurons is decreased in MVP+/- mice after extended MD, suggesting a reduction of functional synapses. Correspondingly, upregulation of surface GluA1 AMPA receptors is reduced in MVP+/- mice after extended MD, and is accompanied by altered expression of STAT1 and phosphorylated ERK, which have been previously implicated in OD plasticity. Normalization of STAT1 levels by introducing STAT1 shRNA rescues surface GluA1 and open-eye responses, implicating STAT1 as a downstream effector of MVP. These findings demonstrate a specific role for MVP as a key molecule influencing the homeostatic component of activity-dependent synaptic plasticity, and potentially the corresponding phenotypes of 16p11.2 microdeletion syndrome. Keywords: autism spectrum disorder; glutamate receptors; ocular dominance plasticity; signaling; molecules; synapse development; visual cortexNational Institutes of Health (U.S.) (Grant MH085802)National Institutes of Health (U.S.) (Grant EY007023