Sudhir Sahasrabudhe

A protein interaction network of the malaria parasite Plasmodium falciparum

Plasmodium falciparum causes the most severe form of malaria and kills up to 2.7 million people annually. Despite the global importance of P. falciparum, the vast majority of its proteins have not been characterized experimentally. Here we identify P. falciparum protein–protein interactions using a high-throughput version of the yeast two-hybrid assay that circumvents the difficulties in expressing P. falciparum proteins in Saccharomyces cerevisiae. From more than 32,000 yeast two-hybrid screens with P. falciparum protein fragments, we identified 2,846 unique interactions, most of which include at least one previously uncharacterized protein. Informatic analyses of network connectivity, coexpression of the genes encoding interacting fragments, and enrichment of specific protein domains or Gene Ontology annotations were used to identify groups of interacting proteins, including one implicated in chromatin modification, transcription, messenger RNA stability and ubiquitination, and another implicated in the invasion of host cells. These data constitute the first extensive description of the protein interaction network for this important human pathogen.

RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels

Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS–RAF–MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)—a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS–RAF–MEK pathway.

A human MAP kinase interactome

Mitogen-activated protein kinase (MAPK) pathways form the backbone of signal transduction in the mammalian cell. Here we applied a systematic experimental and computational approach to map 2,269 interactions between human MAPK-related proteins and other cellular machinery and to assemble these data into functional modules. Multiple lines of evidence including conservation with yeast supported a core network of 641 interactions. Using small interfering RNA knockdowns, we observed that approximately one-third of MAPK-interacting proteins modulated MAPK-mediated signaling. We uncovered the Na-H exchanger NHE1 as a potential MAPK scaffold, found links between HSP90 chaperones and MAPK pathways and identified MUC12 as the human analog to the yeast signaling mucin Msb2. This study makes available a large resource of MAPK interactions and clone libraries, and it illustrates a methodology for probing signaling networks based on functional refinement of experimentally derived protein-interaction maps.

A Protein Interaction Network for Ecm29 Links the 26 S Proteasome to Molecular Motors and Endosomal Components

Ecm29 is a 200-kDa HEAT repeat protein that binds the 26 S proteasome. Genome-wide two-hybrid screens and mass spectrometry have identified molecular motors, endosomal components, and ubiquitin-proteasome factors as Ecm29-interacting proteins. The C-terminal half of human Ecm29 binds myosins and kinesins; its N-terminal region binds the endocytic proteins, Vps11, Rab11-FIP4, and rabaptin. Whereas full-length FLAG-Ecm29, its C-terminal half, and a small central fragment of Ecm29 remain bound to glycerol-gradient-separated 26 S proteasomes, the N-terminal half of Ecm29 does not. Confocal microscopy showed that Ecm-26 S proteasomes are present on flotillin-positive endosomes, but they are virtually absent from caveolin- and clathrin-decorated endosomes. Expression of the small central fragment of Ecm29 markedly reduces proteasome association with flotillin-positive endosomes. Identification of regions within Ecm29 capable of binding molecular motors, endosomal proteins, and the 26 S proteasome supports the hypothesis that Ecm29 serves as an adaptor for coupling 26 S proteasomes to specific cellular compartments.

Interaction of an atypical Plasmodium falciparum ETRAMP with human apolipoproteins

BackgroundIn order to establish a successful infection in the human host, the malaria parasite Plasmodium falciparum must establish interactions with a variety of human proteins on the surface of different cell types, as well as with proteins inside the host cells. To better understand this aspect of malaria pathogenesis, a study was conducted with the goal of identifying interactions between proteins of the parasite and those of its human host.MethodsA modified yeast two-hybrid methodology that preferentially selects protein fragments that can be expressed in yeast was used to conduct high-throughput screens with P. falciparum protein fragments against human liver and cerebellum libraries. The resulting dataset was analyzed to exclude interactions that are not likely to occur in the human host during infection.ResultsAn initial set of 2,200 interactions was curated to remove proteins that are unlikely to play a role in pathogenesis based on their annotation or localization, and proteins that behave promiscuously in the two-hybrid assay, resulting in a final dataset of 456 interactions. A cluster that implicates binding between P. falciparum PFE1590w/ETRAMP5, a putative parasitophorous vacuole membrane protein, and human apolipoproteins ApoA, ApoB and ApoE was selected for further analysis. Different isoforms of ApoE, which are associated with different outcomes of malaria infection, were shown to display differential interactions with PFE1590w.ConclusionA dataset of interactions between proteins of P. falciparum and those of its human host was generated. The preferential interaction of the P. falciparum PFE1590w protein with the human ApoE ε3 and ApoE ε4 isoforms, but not the ApoE ε2 isoform, supports the hypothesis that ApoE genotype affects risk of malaria infection. The dataset contains other interactions of potential relevance to disease that may identify possible vaccine candidates and drug targets.

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

BackgroundA newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma.MethodsUsing a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds.ResultsAmong 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo.ConclusionsThis staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.

Abstract 2543: PRLX 93936, is a potent, novel, tumor-selective, small molecule with a unique mechanism of action

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Currently in Phase I testing, PRLX 93936 was developed and characterized at Prolexys Pharmaceuticals. It is a potent small molecule drug that shows robust and selective anti-cancer activity in a wide array of solid tumors including those with mutations that activate the RAS pathway. PRLX 93936 was chosen as a clinical lead as a result of extensive medicinal chemistry efforts and pharmacological profiling of over 400 novel small molecules. These compounds were designed to cover the chemical space originating from a molecule, Erastin, identified in a screen against isogenic cell lines differentially expressing genes such as those that activate the RAS pathway (Dolma et. al, Cancer Cell 3: 285-296, 2003). Cells that are sensitive to PRLX 93936 undergo caspase dependent apoptosis, necrosis, cell cycle arrest, ion flux and cell shrinkage leading to death and tumor growth inhibition. Characterization of drug treated cells shows that selective activation of RAS and JNK pathway members correlates with compound sensitivity. Research to elucidate the precise mechanism(s) and target(s) through which these biological responses are achieved has revealed that PRLX 93936s mechanism of action is unique. Several key lines of evidence support this conclusion including: 1. COMPARE analysis of PRLX 93936 activity in the NCI 60 panel of cell lines which showed that it is functionally distinct among the agents tested, 2. Profiling studies which examined PRLX 93936 binding or inhibition of enzyme activity against CYP450 enzymes, cell surface receptors and action potential mediating cardiac ion channels revealed only Cyp 3A4 and hERG as potential targets, 3. Kinase profiling of PRLX 93936 against evolutionarily diverse enzymes which identified no significant inhibitory activity, and 4. Genotyping and gene expression profiling studies which have yet to reveal a clear target profile correlation. Since the most common target classes were eliminated as potential suspects, additional non-traditional enzyme targets were screened for relevance to PRLX 93936 activity. Proteomics experiments identified the mitochondrial protein VDAC (Voltage Dependent Anion Channel) as a specific binding protein and potential molecular target of PRLX 93936 (Yagoda et al., Nature. 447: 865-869, 2007). More recent findings indicate that PRLX 93936 is a potent, selective inhibitor of several RAS pathway proteins including key enzymes which regulate the phosphorylation and activity of proteins involved in the RAS/JNK pathway. RNAi based validation studies and synergistic anti-cancer activity observed in combination with specific targeted drugs support these findings. PRLX 93936 may therefore represent a novel class of cancer therapeutics that target the majority of tumors characterized by RAS pathway activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2543.