Structure and catalytic mechanism of human protein tyrosine phosphatome

Together with protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs) serve as hallmarks in cellular signal transduction by controlling the reversible phosphorylation of their substrates. The human genome is estimated to encode more than 100 PTPs, which can be divided into eleven sub-groups according to their structural and functional characteristics. All the crystal structures of catalytic domains of sub-groups have been elucidated, enabling us to understand their precise catalytic mechanism and to compare their structures across all sub-groups. In this review, I describe the structure and mechanism of catalytic domains of PTPs in the structural context. [BMB Reports 2012; 45(12): 693-699]     

Quantitative proteomic assessment of very early cellular signaling events

Technical limitations have prevented proteomic analyses of events occurring less than 30 s after signal initiation. We developed an automated, continuous quench-flow system allowing quantitative proteomic assessment of very early cellular signaling events (qPACE) with a time resolution of 1 s. Using this technique, we determined that autophosphorylation of the epidermal growth factor receptor occurs within 1 s after ligand stimulation and is followed rapidly by phosphorylation of the downstream signaling intermediates Src homologous and collagen-like protein and phospholipase C gamma 1.     

A proteomic assessment of muscle contractile alterations during unloading and reloading

Unloading of skeletal muscle causes atrophy and altered contractility. To identify major muscle proteins responding significantly to the altered loading and to elucidate how the contractile alterations reflect potential proteomic modifications, we examined protein expression in the rat soleus muscle during 3-week hindlimb suspension and 2-week reloading. Compared with unsuspended controls, experimental animals had a 0.5- to 0.6-fold decrease in tension during unloading and early reloading, comparable to 0.2- to 0.6-fold decreases in the protein levels of myosin light chain 1 (MLC1), alpha-actin, tropomyosin beta-chain, and troponins T1 and T2. The observed 1.4- to 1.6-fold increase in shortening velocity appears to reflect 1.2- to 9.0-fold increases in the protein levels of fast-type MLC2, glycolytic enzymes, and creatine kinase, and 0.2- to 0.3-fold decreases in slow-type troponins T1 and T2. The levels of three heat shock proteins (p20, alpha crystallin B chain, and HSP90) decreased during unloading but returned to control levels during reloading. These results imply that proteomic responses to unloading change overall myofibrillar integrity and metabolic regulation, resulting in altered contractility.     

Assessment of protein-tyrosine phosphatase 1B substrate specificity using "inverse alanine scanning"

An "inverse alanine scanning" peptide library approach has been developed to assess the substrate specificity of protein-tyrosine phosphatases (PTPases). In this method each Ala moiety in the parent peptide, Ac-AAAApYAAAA-NH(2), is separately and sequentially replaced by the 19 non-Ala amino acids to generate a library of 153 well defined peptides. The relatively small number of peptides allows the acquisition of explicit kinetic data for all library members, thereby furnishing information about the contribution of individual amino acids with respect to substrate properties. The approach was applied to protein-tyrosine phosphatase 1B (PTP1B) as a first example, and the highly potent peptide substrate Ac-ELEFpYMDYE-NH(2) (k(cat)/K(m) 2.2 +/- 0.05 x 10(7) M(-1) s(-1)) has been identified. More importantly, several heretofore unknown features of the substrate specificity of PTP1B were revealed. This includes the ability of PTP1B to accommodate acidic, aromatic, and hydrophobic residues at the -1 position, a strong nonpreference for Lys and Arg residues in any position, and the first evidence that residues well beyond the +1 position contribute to substrate efficacy.     

Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.     

Integration of multidimensional chromatographic protein separations with a combined "top-down" and "bottom-up" proteomic strategy

In this paper, we present a combined top-down/bottom-up proteomic analysis workflow for the characterization of proteomic samples. This workflow combines protein fractionation (multidimensional chromatographic separation) with parallel online ESI-TOF-MS intact protein analysis, and fraction collection. Collected fractions were digested and protein identifications were produced using MALDI Q-TOF-MS analysis. These identifications were then linked with corresponding ESI-TOF-MS intact protein mass data to permit full protein characterization. This methodology was applied to an E. coli cytosolic protein fraction, and enabled the identification and characterization of proteins exhibiting co-translational processing, post-translational modification, and proteolytic processing events. The approach also provided the ability to distinguish between closely related protein isoforms. The summary of results from this study indicated that roughly one-third of all detected components generated corresponding data from both top-down and bottom-up analyses, and that significant and novel information can be derived from this application of the hybrid analytical methodology.     

A longitudinal proteomic assessment of peptide degradation and loss under acidic storage conditions

Sample preparation prior to analysis by liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) usually involves the storage of frozen peptide samples in an acidic environment for variable time periods. Questions arose in our laboratory regarding the stability of peptides in acid under medium- to long-term storage. Thus, a 10-month longitudinal study was designed to assess the effect on storage of tryptic peptides at −20 and −80 °C under acidic conditions. Our conclusion and proposal from this evaluation is that the optimal storage conditions of peptide samples in acid for proteomic experiments is at −80 °C and, ideally, as separate aliquots.     

Global survey of organ and organelle protein expression in mouse: combined proteomic and transcriptomic profiling

Organs and organelles represent core biological systems in mammals, but the diversity in protein composition remains unclear. Here, we combine subcellular fractionation with exhaustive tandem mass spectrometry-based shotgun sequencing to examine the protein content of four major organellar compartments (cytosol, membranes [microsomes], mitochondria, and nuclei) in six organs (brain, heart, kidney, liver, lung, and placenta) of the laboratory mouse, Mus musculus. Using rigorous statistical filtering and machine-learning methods, the subcellular localization of 3274 of the 4768 proteins identified was determined with high confidence, including 1503 previously uncharacterized factors, while tissue selectivity was evaluated by comparison to previously reported mRNA expression patterns. This molecular compendium, fully accessible via a searchable web-browser interface, serves as a reliable reference of the expressed tissue and organelle proteomes of a leading model mammal.     

Biotinylated anisomycin: a comparison of classical and "click" chemistry approaches

Two approaches to the synthesis of biotinylated derivatives of the stress-activated protein kinase (SAPK) pathway activator anisomycin have been investigated. Attachment of the biotin moiety to the central core was achieved either through the use of a classical displacement reaction on alpha-halo carbonyl derivatives of biotin or through a copper(I)-catalyzed 1,3-dipolar Huisgen cycloaddition ("click") coupling of biotinylated azides to propargyl-marked analogues of anisomycin. In each case, the resultant N-linked molecular probes were found to be active in SAPK pathway immunoblot assays, while their O-linked counterparts were inactive. However, in sharp contrast to the classical coupling approach which results in low coupling yields, the aqueous "click" coupling process was found to deliver high yields of biotinylated probes, making it the conjugation method of choice. A survey of the available methods for the addition of a propargyl marker onto a range of chemical functionalities strongly suggests that this copper(I)-catalyzed 1,3-dipolar Huisgen cycloaddition approach to biotinylation may be generally applied.     

Biosynthesis and control of beta-lactam antibiotics: the early steps in the "classical" tripeptide pathway

The interaction between growth and secondary metabolism develops from physiological responses of the producer organism to its environment. Nutrients are channelled into primary growth processes or into secondary processes such as antibiotic biosynthesis by a variety of metabolic controls, the nature of which has been extensively studied in organisms producing beta-lactam antibiotics via the tripeptide, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine. In the following article we review the early stages of beta-lactam biosynthesis in fungi and actinomycetes, keeping in mind the regulation of primary pathways that provide the amino acid precursors of this group of antibiotics, as well as the regulation of the secondary pathway itself. Of special importance to organisms engaging in secondary metabolism are the control mechanisms that suppress the nonessential process during rapid growth but allow secondary metabolic genes to be expressed and resources to be diverted when environmental factors generate the appropriate biochemical signals.     

Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.     

Global assessment of cross-hybridization for oligonucleotide arrays.

Cross-hybridization is the tendency for chains of nucleic acids to bind to other chains of nucleic acids that have similar but not identical sequences. This has the potential to make the interpretation of microarray experiments difficult since intensity at a spot on the array does not simply depend on the quantity of target in the sample. We propose a method for evaluating the extent of cross-hybridization in oligonucleotide arrays for data arising from a typical microarray experiment. We show that the level of cross-hybridization can be quite substantial. We argue that this makes the interpretation of the difference calls provided by MAS 5.0 difficult.     

Src protein-tyrosine kinase structure and regulation

Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPalpha displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the alphaD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu(4)Tyr).     

Homoassociation of VE-cadherin follows a mechanism common to "classical" cadherins

Vascular endothelial cadherin (VE-cadherin/cadherin5) is specifically expressed in adherens junctions of endothelial cells and exerts important functions in cell-cell adhesion as well as signal transduction. To analyze the mechanism of VE-cadherin homoassociation, the ectodomains CAD1-5 were connected by linker sequences to the N terminus of the coiled-coil domain of cartilage matrix protein (CMP). The chimera VECADCMP were expressed in mammalian cells. The trimeric coiled-coil domain leads to high intrinsic domain concentrations and multivalency promoting self-association. Ca(2+)-dependent homophilic association of VECADCMP was detected in solid phase assays and cross-linking experiments. A striking analogy to homoassociation of type I ("classical") cadherins like E, N or P-cadherin was observed when interactions in VECADCMP and between these trimeric proteins were analyzed by electron microscopy. Ca(2+)-dependent ring-like and double ring-like arrangements suggest interactions between domains 1 and 2 of the ectodomains, which may be correlated with lateral and adhesive contacts in the adhesion process. Association to complexes composed of two VECADCMP molecules was also demonstrated by chemical cross-linking. No indication for an antiparallel association of VECAD ectodomains to hexameric complexes as proposed by Legrand et al. was found. Instead the data suggest that homoassociation of VE-cadherin follows the conserved mechanism of type I cadherins.     

Effects of oral vitamin C supplementation in hemodialysis patients: a proteomic assessment

Evidence indicates that oxidative stress is present in dialysis patients, and is associated with vitamin C deficiency. Limited data are available regarding the effects of vitamin C supplementation on oxidative stress and inflammation markers in these patients. Moreover, there are no data available on plasma polypeptide fingerprints by proteome analysis before and after vitamin C supplementation. Therefore, we analyzed plasma samples from a prospective, randomized, open-labeled trial to assess the effects of oral vitamin C supplementation (250 mg three times per week), to define the plasma polypeptide pattern in hemodialysis patients. Our results reveal that more than 30 polypeptides show significant changes in the dialysis patients in comparison to controls with normal renal function, and that several polypeptides are affected/normalized by oral vitamin C supplementation. These results underline the remarkable potential for proteomics to recognize specific peptide profiles in different pathological situations, which might not be detected by classical methods.     

Ethological validation and the assessment of anxiety-like behaviours: methodological comparison of classical analyses and structural approaches

The research on emotional reactivity usually implies the use of standardised behavioural tests that provide a quick idea of the effect of a treatment on the reactivity of subjects to potentially dangerous situations. Many validity criteria have been considered to evaluate these tests. This validity concept supports the idea that animals' behaviour in these tests model human anxiety. Generally, those criteria repeatedly labelled as "ethological validation" refer to the analogy between animals and human in the meaning of the test situation. Although the content of the ethological validation concept is heterogeneous, it is steadily related to a fixed interpretation of the behavioural items produced in a given experimental setting. The basic assumption of such reasoning is that the behavioural items would always be expressed in the same behavioural context whatever the subject, its gender, strain or species, thoroughly asserting a predefined subjective state. Using multivariate and textual analysis, we found evidence that the "ethological validation" recourse to an a priori interpretation for a given behavioural variable may be deceptive. We defend the idea that the meaning of a behavioural variable should be restricted to the general context where it arose. Theoretical propositions and methodological options are discussed.