Integrin-linked kinase is a functional Mn2+-dependent protein kinase that regulates glycogen synthase kinase-3β (GSK-3beta) phosphorylation.

Background: Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain.

An integrated global strategy for cell lysis, fractionation, enrichment and mass spectrometric analysis of phosphorylated peptides.

Recently, the field of phosphoproteomics has progressed to the point where thousands of protein phosphorylations can be analyzed simultaneously and used to address significant biological questions. However, several challenges still exist in current LC-MS/MS-based phosphoproteomics methods. Among these are the increased dynamic range of phosphoproteomics samples (due to low stoichiometry of most protein phosphorylations), insufficient inhibition of phosphatase activity, and neutral losses which occur during phosphopeptide fragmentation by MS(n).

Ecological adaptation of diverse honey bee (Apis mellifera) populations.

Background: Honey bees are complex eusocial insects that provide a critical contribution to human agricultural food production. Their natural migration has selected for traits that increase fitness within geographical areas, but in parallel their domestication has selected for traits that enhance productivity and survival under local conditions. Elucidating the biochemical mechanisms of these local adaptive processes is a key goal of evolutionary biology.

Development of a Chlamydia trachomatis T cell Vaccine.

The immune correlates of protection for most of the currently used vaccines are based on long-lived humoral immunity. Vaccines based on humoral immunity alone are unlikely to protect against infections caused by intracellular pathogens and today's most pressing infectious diseases of public health importance are caused by intracellular infections that not only include Chlamydia trachomatis but also tuberculosis, malaria, and HIV/AIDS. For these infections, vaccines that induce cellular immune responses are essential.

Release of membrane-bound vesicles and inhibition of tumor cell adhesion by the peptide Neopetrosiamide A.

Background: Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown.

Methodology/principal Findings: We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of beta1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium.

PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes.

Motivation: PSORTb has remained the most precise bacterial protein subcellular localization (SCL) predictor since it was first made available in 2003. However, the recall needs to be improved and no accurate SCL predictors yet make predictions for archaea, nor differentiate important localization subcategories, such as proteins targeted to a host cell or bacterial hyperstructures/organelles. Such improvements should preferably be encompassed in a freely available web-based predictor that can also be used as a standalone program.

Pseudopodial actin dynamics control epithelial-mesenchymal transition in metastatic cancer cells.

A key cellular process associated with the invasive or metastatic program in many cancers is the transformation of epithelial cells toward a mesenchymal state, a process called epithelial to mesenchymal transition or EMT. Actin-dependent protrusion of cell pseudopodia is a critical element of mesenchymal cell migration and therefore of cancer metastasis. However, whether EMT occurs in human cancers and, in particular, whether it is a prerequisite for tumor cell invasion and metastasis, remains a subject of debate.

The pathogenic E. coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling.

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are diarrhoeal pathogens that cause the formation of attaching and effacing (A/E) lesions on infected host cells. These pathogens encode a type III secretion system (T3SS) used to inject effector proteins directly into host cells, an essential requirement for virulence. In this study, we identified a function for the type III secreted effector EspZ.

Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products.

Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis.

An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages.

Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells.

Structural and biochemical characterization of SrcA, a multi-cargo type III secretion chaperone in Salmonella required for pathogenic association with a host.

Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known.

Quantitative analysis of proteome coverage and recovery rates for upstream fractionation methods in proteomics.

The proteome of any cell or even any subcellular fraction remains too complex for complete analysis by one dimension of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hence, to achieve greater depth of coverage for a proteome of interest, most groups routinely subfractionate the sample prior to LC-MS/MS so that the material entering LC-MS/MS is less complex than the original sample. Protein and/or peptide fractionation methods that biochemists have used for decades, such as strong cation exchange chromatography (SCX), isoelectric focusing (IEF) and SDS-PAGE, are the most common prefractionation methods used currently.

A comprehensive proteomic analysis of the type III secretome of Citrobacter rodentium.

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease.

MSQuant, an open source platform for mass spectrometry-based quantitative proteomics.

Mass spectrometry-based proteomics critically depends on algorithms for data interpretation. A current bottleneck in the rapid advance of proteomics technology is the closed nature and slow development cycle of vendor-supplied software solutions. We have created an open source software environment, called MSQuant, which allows visualization and validation of peptide identification results directly on the raw mass spectrometric data.

Sequestosome-1/p62 is the key intracellular target of innate defense regulator peptide.

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1.

Phosphoproteomics--finally fulfilling the promise?

Networks of protein-protein and protein-metabolite interactions are commonly found in biological systems where signals must be passed from one location or component within a cell to another, such as from a receptor on the plasma membrane to a transcription factor in the nucleus. Regulation of such networks, or signal transduction pathways, is often achieved by transient, reversible modification of the components involved. Several types of post-translational modifications of proteins are employed in signal transduction including ubiquitylation of lysines and palmitoylation of cysteines, but by far the best appreciated and apparently the most important involves phosphorylation of serine, threonine and tyrosine residues.

The innate immune and systemic response in honey bees to a bacterial pathogen, Paenibacillus larvae.

Background: There is a major paradox in our understanding of honey bee immunity: the high population density in a bee colony implies a high rate of disease transmission among individuals, yet bees are predicted to express only two-thirds as many immunity genes as solitary insects, e.g., mosquito or fruit fly.

Intracellular receptor for human host defense peptide LL-37 in monocytes.

The human cationic host defense peptide LL-37 has a broad range of immunomodulatory, anti-infective functions. A synthetic innate defense regulator peptide, innate defense regulator 1 (IDR-1), based conceptually on LL-37, was recently shown to selectively modulate innate immunity to protect against a wide range of bacterial infections. Using advanced proteomic techniques, ELISA, and Western blotting procedures, GAPDH was identified as a direct binding partner for LL-37 in monocytes.

Contributions of quantitative proteomics to understanding membrane microdomains.

Membrane microdomains, e.g., lipid rafts and caveolae, are crucial cell surface organelles responsible for many cellular signaling and communication events, which makes the characterization of their proteomes both interesting and valuable.

Cell-based identification of natural substrates and cleavage sites for extracellular proteases by SILAC proteomics.

Proteolysis is one of the most important post-translational modifications of the proteome with every protein undergoing proteolysis during its synthesis and maturation and then upon inactivation and degradation. Extracellular proteolysis can either activate or inactivate bioactive molecules regulating physiological and pathological processes. Therefore, it is important to develop non-biased high-content screens capable of identifying the substrates for a specific protease.

Identification of protein complexes with quantitative proteomics in S. cerevisiae.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.

Moving closer to the lipid raft proteome using quantitative proteomics.

Lipid rafts are small subdomains of the plasma membrane enriched in cholesterol, sphingolipids, saturated phospholipids and specific proteins. They are thought to act as coordination centres for signal transduction pathways so their protein composition is of particular biological interest. Rafts are refractory to solubilization in non-ionic detergents so they can be biochemically enriched by floatation on a sucrose density gradient but several other membranes co-migrate with rafts in such a procedure.

Mitochondria do not contain lipid rafts, and lipid rafts do not contain mitochondrial proteins.

Lipid rafts are membrane microdomains involved in many cellular functions, including transduction of cellular signals and cell entry by pathogens. Lipid rafts can be enriched biochemically by extraction in a nonionic detergent at low temperature, followed by floatation on a sucrose density gradient. Previous proteomic studies of such detergent-resistant membranes (DRMs) are in disagreement about the presence of mitochondrial proteins in raft components.

Changes in protein expression during honey bee larval development.

Background: The honey bee (Apis mellifera), besides its role in pollination and honey production, serves as a model for studying the biochemistry of development, metabolism, and immunity in a social organism. Here we use mass spectrometry-based quantitative proteomics to quantify nearly 800 proteins during the 5- to 6-day larval developmental stage, tracking their expression profiles.

Results: We report that honey bee larval growth is marked by an age-correlated increase of protein transporters and receptors, as well as protein nutrient stores, while opposite trends in protein translation activity and turnover were observed.

Biochemical and proteomic approaches for the study of membrane microdomains.

Many cellular signaling and communication events take place at the plasma membrane and thus the characterization of the plasma membrane proteome has been a hot research area in the hopes of learning more about these processes. Membrane microdomains are large protein and lipid complexes found on the cell surface membrane, able to concentrate or recruit signaling molecules or factors. The first step of any organelle proteomics study is to get a pure and enriched protein sample yet this has always been problematic in membrane proteomics as it is virtually impossible to purify a specific membrane type to homogeneity.