Phosphorylation of serine palmitoyltransferase long chain-1 (SPTLC1) on tyrosine 164 inhibits its activity and promotes cell survival.

In BCR-ABL-expressing cells, sphingolipid metabolism is altered. Because the first step of sphingolipid biosynthesis occurs in the endoplasmic reticulum (ER), our objective was to identify ABL targets in the ER. A phosphoproteomic analysis of canine pancreatic ER microsomes identified 49 high scoring phosphotyrosine-containing peptides. These were then categorized in silico and validated in vitro. We demonstrated that the ER-resident human protein serine palmitoyltransferase long chain-1 (SPTLC1), which is the first enzyme of sphingolipid biosynthesis, is phosphorylated at Tyr(164) by the tyrosine kinase ABL. Inhibition of BCR-ABL using either imatinib or shRNA-mediated silencing led to the activation of SPTLC1 and to increased apoptosis in both K562 and LAMA-84 cells. Finally, we demonstrated that mutation of Tyr(164) to Phe in SPTLC1 increased serine palmitoyltransferase activity. The Y164F mutation also promoted the remodeling of cellular sphingolipid content, thereby sensitizing K562 cells to apoptosis. Our observations provide a mechanistic explanation for imatinib-mediated cell death and a novel avenue for therapeutic strategies.

MAPK scaffolding by BIT1 in the Golgi complex modulates stress resistance.

The endoplasmic reticulum (ER) is an essential organelle whose major functions are to ensure proper secretory protein folding and trafficking. These mechanisms involve the activation of specific ER-resident molecular machines, which might be regulated by their membranous environments. Based on this observation, we aimed to characterize the proteome of ER-membrane microdomains to identify new components of the ER that have a role in secretory pathway-associated functions. Using this approach with dog pancreatic rough microsomes, we found that mitochondrial Bcl-2 inhibitor of transcription (BIT1) localized in the early secretory pathway and accumulated in the Golgi complex. Using both a chimeric protein of the luminal and transmembrane domains of ER-resident TRAPalpha and the cytosolic domain of BIT1, and silencing of BIT1 expression, we perturbed endogenous BIT1 oligomerization and localization to the Golgi. This led to enhanced ERK signaling from the Golgi complex, which resulted in improved stress resistance. This work provides the first evidence for the existence of ER microdomains that are involved in the regulation of BIT1 structure and trafficking, and identifies BIT1 as a negative regulator of the ERK-MAPK signaling pathway in the Golgi.

A community standard format for the representation of protein affinity reagents.

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site.

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome.

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Integrating forward and reverse proteomics to unravel protein function.

To date, proteomics approaches have aimed to either identify novel proteins or change in protein expression/modification in various organisms under normal or disease conditions. One major aspect of functional proteomics is to identify protein biological properties in a given context, however, forward proteomics approaches alone cannot complete this goal. Indeed, with the increasing successes of such proteomics-based research strategies and the subsequent increasing amounts of proteins identified with unknown molecular functions, approaches allowing for systematic analyses of protein functions are desired. In this review, we propose to depict the complementarities of forward and reverse proteomics approaches in the definite understanding of protein functions. This dual strategy requires a data integration loop which allows for systematic characterization of protein function(s). The details of the integrative process combining both in silico and experimental resources and tools are presented. Altogether, we believe that the integration of forward and reverse proteomics approaches supported by bioinformatics will provide an efficient path towards systems biology.