Proteomic Analysis of Signaling Network Regulation in Renal Cell Carcinomas with Differential Hypoxia-Inducible Factor-2α Expression

Background

The loss of von Hippel–Lindau (VHL) protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of VHL in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2α (HIF2α) instead of hypoxia-inducible factor-1α (HIF1α).

Methods and Principal Findings

We used renal carcinoma cell lines that recapitulate the differences between mutant VHL and wild-type VHL genotypes. Utilizing a method relying on extracted peptide intensities as a label-free approach for quantitation by liquid chromatography–mass spectrometry, our proteomics study revealed regulation of key proteins important for cancer cell survival, proliferation and stress-resistance, and implicated differential regulation of signaling networks in VHL-mutant renal cell carcinoma. We also observed upregulation of cellular energy pathway enzymes and the stress-responsive mitochondrial 60-kDa heat shock protein. Finding reliance on glutaminolysis in VHL-mutant renal cell carcinoma was of particular significance, given the generally predominant dependence of tumors on glycolysis. The data have been deposited to the ProteomeXchange with identifier PXD000335.

Conclusions and Significance

Pathway analyses provided corroborative evidence for differential regulation of molecular and cellular functions influencing cancer energetics, metabolism and cell proliferation in renal cell carcinoma with distinct VHL genotype. Collectively, the differentially regulated proteome characterized by this study can potentially guide translational research specifically aimed at effective clinical interventions for advanced VHL-mutant, HIF2α-over-expressing tumors.     

Analysis of dynamic tyrosine phosphoproteome in LFA-1 triggered migrating T-cells

The ordered, directional migration of T-lymphocytes is a key process during immune surveillance and response. This requires cell adhesion to the high endothelial venules or to the extracellular matrix by a series of surface receptor/ligand interactions involving adhesion molecules of the integrin family including lymphocyte function associated molecule-1 (LFA-1) and intercellular adhesion molecules (ICAMs). Reversible protein phosphorylation is emerging as a key player in the regulation of biological functions with tyrosine phosphorylation playing a crucial role in signal transduction. Thus, the study of this type of post-translational modification at the proteomic level has great biological significance. In this work, phospho-enriched cell lysates from LFA-1-triggered migrating human T-cells were subjected to immunoaffinity purification of tyrosine phosphorylated proteins, mass spectrometric, and bioinformatic analysis. In addition to the identification of several well-documented proteins, the analysis suggested involvement of a number of new and novel proteins in LFA-1 induced T-cell migration. This dataset expands the list of the signaling components of the LFA-1 induced phosphotyrosine protein complexes in migrating T-cells that will be extremely useful in the study of their specific roles within LFA-1 associated signaling pathways. Identification of proteins previously not reported in the context of LFA-1 stimulated signal transduction might provide new insights into understanding the LFA-1 signaling networks and aid in the search for new potential therapeutic targets.     

Detection and identification of 4‐hydroxy‐2‐nonenal Schiff‐base adducts along with products of Michael addition using data‐dependent neutral loss‐driven MS3 acquisition: Method evaluation through an in vitro study on cytochrome c oxidase modifications

We report a data‐dependent neutral‐loss‐driven MS³ acquisition to enhance, in addition to abundant Michael adducts, the detection of Schiff‐base adducts of proteins and 4‐hydroxy‐2‐nonenal, a reactive end product of lipid peroxidation. In vitro modification of cytochrome c oxidase, a mitochondrial protein complex, was used as a model to evaluate the method. The technique allowed for a confident validation of modification sites and also identified a Schiff‐base adduct in subunit Vb of the protein complex.     

Miltefosine induces apoptosis in arsenite-resistant Leishmania donovani promastigotes through mitochondrial dysfunction

The control of leishmaniasis in absence of vaccine solely depends on the choice of chemotherapy. The major hurdle in successful leishmanial chemotherapy is emergence of drug resistance. Miltefosine, the first orally administrable anti-leishmanial drug, has shown the potential against drug-resistant strains of Leishmania. However, there are discrepancies regarding the involvement of P-glycoprotein (Pgp) and sensitivity of miltefosine in multiple drug-resistant (MDR) cell lines that overexpress Pgp in Leishmania. To address this, the effect of miltefosine in arsenite-resistant Leishmania donovani (Ld-As20) promastigotes displaying an MDR phenotype and overexpressing Pgp-like protein was investigated in the current study. Results indicate that Ld-As20 is sensitive to miltefosine. Miltefosine induces process of programmed cell death in Ld-As20 in a time-dependent manner as determined by cell shrinkage, externalization of phosphatidylserine and DNA fragmentation. Miltefosine treatment leads to loss of mitochondrial membrane potential and the release of cytochrome C with consequent activation of cellular proteases. Activation of cellular proteases resulted in activation of DNase that damaged kinetoplast DNA and induced dyskinetoplasty. These data indicate that miltefosine causes apoptosis-like death in arsenite-resistant L. donovani.     

Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli

Low yields of recombinant expression represent a major barrier to the physical characterization of membrane proteins. Here, we have identified genes that globally enhance the production of properly folded G protein-coupled receptors (GPCRs) in Escherichia coli. Libraries of bacterial chromosomal fragments were screened using two separate systems that monitor: (i) elevated fluorescence conferred by enhanced expression of GPCR-GFP fusions and (ii) increased binding of fluorescent ligand in cells producing more active receptor. Three multi-copy hits were isolated by both methods: nagD, encoding the ribonucleotide phosphatase NagD; a fragment of nlpD, encoding a truncation of the predicted lipoprotein NlpD, and the three-gene cluster ptsN-yhbJ-npr, encoding three proteins of the nitrogen phosphotransferase system. Expression of these genes resulted in a 3- to 10-fold increase in the yields of different mammalian GPCRs. Our data is consistent with the hypothesis that the expression of these genes may serve to maintain the integrity of the bacterial periplasm and to provide a favorable environment for proper membrane protein folding, possibly by inducing a fine-tuned stress response and/or via modifying the composition of the bacterial cell envelope.     

Genetic variation in histidine rich proteins among Indian Plasmodium falciparum population: possible cause of variable sensitivity malaria of rapid diagnostic tests

ABSTRACT: BACKGROUND: Rapid diagnostic tests (RDTs) have revolutionized the diagnosis of malaria. Among the various factors affecting RDTs sensitivity is genetic variation of the antigen used. The genetic variation in PfHRP2 and PfHRP3 proteins was studied among the Indian Plasmodium falciparum isolates. METHODS: One hundred and forty isolates of P. falciparum were collected from six geographical regions of India. Target genes encoding PfHRP2 and PfHRP3 antigens were sequenced to study genetic polymorphism. Minimum detection limit giving a positive rapid diagnostic test was also determined. RESULTS: Extensive variations were observed in amino acid repeat types of PfHRP2 and PfHRP3. PfHRP2 exhibited more polymorphism than PfHRP3. Significant relation was observed between type 2 and type 7 repeats and RDT detection rate as higher number of these repeats showed better sensitivity with RDTs. CONCLUSION: The results provide insights into the genetic diversity of Pfhrp2 and Pfhrp3 genes among Indian P. falciparum population and its relation to RDT sensitivity.     

Genome-wide copy number analysis of single cells

Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the genome-wide copy number analysis of single nuclei isolated from mixed populations of cells. Single-nucleus sequencing (SNS), combines flow sorting of single nuclei on the basis of DNA content and whole-genome amplification (WGA); this is followed by next-generation sequencing to quantize genomic intervals in a genome-wide manner. Multiplexing of single cells is discussed. In addition, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ～3 d from flow cytometry to sequence-ready DNA libraries.