RESUMO
We describe PGTools, an open source software suite for analysis and visualization of proteogenomic data. PGTools comprises applications, libraries, customized databases, and visualization tools for analysis of mass-spectrometry data using combined proteomic and genomic backgrounds. A single command is sufficient to search databases, calculate false discovery rates, group and annotate proteins, generate peptide databases from RNA-Seq transcripts, identify altered proteins associated with cancer, and visualize genome scale peptide data sets using sophisticated visualization tools. We experimentally confirm a subset of proteogenomic peptides in human PANC-1 cells and demonstrate the utility of PGTools using a colorectal cancer data set that led to the identification of 203 novel protein coding regions missed by conventional proteomic approaches. PGTools should be equally useful for individual proteogenomic investigations as well as international initiatives such as chromosome-centric Human Proteome Project (C-HPP). PGTools is available at http://qcmg.org/bioinformatics/PGTools.
Assuntos
Cromossomos Humanos/química , Neoplasias Colorretais/genética , Neoplasias Pancreáticas/genética , Proteômica/estatística & dados numéricos , Software , Linhagem Celular Tumoral , Bases de Dados de Proteínas , Humanos , Espectrometria de Massas , Anotação de Sequência Molecular , Fases de Leitura Aberta , Proteoma/genética , Proteômica/métodos , Análise de Sequência de RNA , TranscriptomaRESUMO
A striking feature of the CLIC (chloride intracellular channel) protein family is the ability of its members to convert between a soluble state and an integral membrane channel form. Direct evidence of the structural transition required for the CLIC protein to autonomously insert into the membrane is lacking, largely because of the challenge of probing the conformation of the membrane-bound protein. However, insights into the CLIC transmembrane form can be gained by biophysical methods such as fluorescence resonance energy transfer (FRET) spectroscopy. This approach was used to measure distances from tryptophan 35, located within the CLIC1 putative N-domain transmembrane region, to three native cysteine residues within the C-terminal domain. These distances were computed both in aqueous solution and upon the addition of membrane vesicles. The FRET distances were used as constraints for modeling of a structure for the CLIC1 integral membrane form. The data are suggestive of a large conformational unfolding occurring between the N- and C-domains of CLIC1 upon interaction with the membrane. Consistent with previous findings, the N-terminal domain of CLIC1 is likely to insert into the lipid bilayer, while the C-domain remains in solution on the extravesicular side of the membrane.
Assuntos
Canais de Cloreto/metabolismo , Proteínas de Membrana/metabolismo , Canais de Cloreto/química , Espectroscopia de Ressonância de Spin Eletrônica , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Humanos , Modelos Moleculares , Ligação Proteica , Espectrometria de Fluorescência , Marcadores de SpinRESUMO
The crystal structures of two CLIC family members DmCLIC and EXC-4 from the invertebrates Drosophila melanogaster and Caenorhabditis elegans, respectively, have been determined. The proteins adopt a glutathione S-transferase (GST) fold. The structures are highly homologous to each other and more closely related to the known structures of the human CLIC1 and CLIC4 than to GSTs. The invertebrate CLICs show several unique features including an elongated C-terminal extension and a divalent metal binding site. The latter appears to alter the ancestral glutathione binding site, and thus, the invertebrate CLICs are unlikely to bind glutathione in the same manner as the GST proteins. Purified recombinant DmCLIC and EXC-4 both bind to lipid bilayers and can form ion channels in artificial lipid bilayers, albeit at low pH. EXC-4 differs from other CLIC proteins in that the conserved redox-active cysteine at the N-terminus of helix 1 is replaced by an aspartic acid residue. Other key distinguishing features of EXC-4 include the fact that it binds to artificial bilayers at neutral pH and this binding is not sensitive to oxidation. These differences with other CLIC family members are likely to be due to the substitution of the conserved cysteine by aspartic acid.
Assuntos
Proteínas de Caenorhabditis elegans/química , Canais de Cloreto/química , Proteínas de Drosophila/química , Animais , Sítios de Ligação , Cátions Bivalentes , Cristalografia por Raios X , Drosophila melanogaster/química , Glutationa , Bicamadas Lipídicas , Metais , Estrutura Terciária de ProteínaRESUMO
Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman's Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.
Assuntos
Interleucina-6/antagonistas & inibidores , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Desenho de Fármacos , Meia-Vida , Humanos , Hibridomas , Interleucina-6/química , Interleucina-6/metabolismo , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-6/química , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade , Células U937RESUMO
Caveolae are cell-surface membrane invaginations that play critical roles in cellular processes including signaling and membrane homeostasis. The cavin proteins, in cooperation with caveolins, are essential for caveola formation. Here we show that a minimal N-terminal domain of the cavins, termed HR1, is required and sufficient for their homo- and hetero-oligomerization. Crystal structures of the mouse cavin1 and zebrafish cavin4a HR1 domains reveal highly conserved trimeric coiled-coil architectures, with intersubunit interactions that determine the specificity of cavin-cavin interactions. The HR1 domain contains a basic surface patch that interacts with polyphosphoinositides and coordinates with additional membrane-binding sites within the cavin C terminus to facilitate membrane association and remodeling. Electron microscopy of purified cavins reveals the existence of large assemblies, composed of a repeating rod-like structural element, and we propose that these structures polymerize through membrane-coupled interactions to form the unique striations observed on the surface of caveolae in vivo.