The Histidine Phosphocarrier Kinase/Phosphorylase from Bacillus Subtilis Is an Oligomer in Solution with a High Thermal Stability

The histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon sources by Gram-positive bacteria. Its X-ray structure, as concluded from crystals of proteins from several species, is a hexamer; however, there are no studies about its conformational stability, and how its structure is modified by the pH. We have embarked on the conformational characterization of HPrK/P of Bacillus subtilis (bsHPrK/P) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, small-angle X-ray-scattering (SAXS) and dynamic light-scattering (DLS)). bsHPrK/P was mainly a hexamer in solution at pH 7.0, in the presence of phosphate. The protein had a high conformational stability, with an apparent thermal denaturation midpoint of ~70 °C, at pH 7.0, as monitored by fluorescence and CD. The protein was very pH-sensitive, precipitated between pH 3.5 and 6.5; below pH 3.5, it had a molten-globule-like conformation; and it acquired a native-like structure in a narrow pH range (between pH 7.0 and 8.0). Guanidinium hydrochloride (GdmCl) denaturation occurred through an oligomeric intermediate. On the other hand, urea denaturation occurred as a single transition, in the range of concentrations between 1.8 and 18 µM, as detected by far-UV CD and fluorescence.     

Regulatory Light Chains in Cardiac Development and Disease

The role of regulatory light chains (RLCs) in cardiac muscle function has been elucidated progressively over the past decade. The RLCs are among the earliest expressed markers during cardiogenesis and persist through adulthood. Failing hearts have shown reduced RLC phosphorylation levels and that restoring baseline levels of RLC phosphorylation is necessary for generating optimal force of muscle contraction. The signalling mechanisms triggering changes in RLC phosphorylation levels during disease progression remain elusive. Uncovering this information may provide insights for better management of heart failure patients. Given the cardiac chamber-specific expression of RLC isoforms, ventricular RLCs have facilitated the identification of mature ventricular cardiomyocytes, opening up possibilities of regenerative medicine. This review consolidates the standing of RLCs in cardiac development and disease and highlights knowledge gaps and potential therapeutic advancements in targeting RLCs.     

Quest for the Crypto-phosphoproteome

Protein phosphorylation is one of the most studied post-translational modifications (PTMs). Despite the remarkable advances in phosphoproteomics, a chemically less-stable subset of the phosphosites, which we call the crypto-phosphoproteome, has remained underexplored due to technological challenges. In this Viewpoint, we briefly summarize the current understanding of these elusive protein phosphorylations and identify the missing pieces for future studies.     

Linking the proteins--elucidation of proteome-scale networks using mass spectrometry

Proteomes are intricate. Typically, thousands of proteins interact through physical association and post-translational modifications (PTMs) to give rise to the emergent functions of cells. Understanding these functions requires one to study proteomes as "systems" rather than collections of individual protein molecules. The abstraction of the interacting proteome to "protein networks" has recently gained much attention, as networks are effective representations, that lose specific molecular details, but provide the ability to see the proteome as a whole. Mostly two aspects of the proteome have been represented by network models: proteome-wide physical protein-protein-binding interactions organized into Protein Interaction Networks (PINs), and proteome-wide PTM relations organized into Protein Signaling Networks (PSNs). Mass spectrometry (MS) techniques have been shown to be essential to reveal both of these aspects on a proteome-wide scale. Techniques such as affinity purification followed by MS have been used to elucidate protein-protein interactions, and MS-based quantitative phosphoproteomics is critical to understand the structure and dynamics of signaling through the proteome. We here review the current state-of-the-art MS-based analytical pipelines for the purpose to characterize proteome-scale networks.     

Regulation of proteins mediating neurodegeneration in experimental autoimmune encephalomyelitis and multiple sclerosis

Multiple sclerosis is characterized by inflammatory demyelination and axonal loss as pathophysiological correlates of relapsing activity and progressive development of clinical disability. The molecular processes involved in this pathogenesis are still unclear as they are quite complex and heterogeneous. In this article we present protein expression analysis of brain and spinal cord tissues from different models of murine experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model for multiple sclerosis. We observed a number of EAE-specific protein expression and PTM differences. Proteome analysis was extended to multiple sclerosis specimens in order to validate the EAE findings. Our findings suggest the regulation of a number of proteins that shed light on the molecular mechanisms of the disease processes taking place in EAE and multiple sclerosis. We found consistent modulation of proteins including serum amyloid P component, sirtuin 2, dihydropyrimidinase-related protein family proteins, stathmin 1, creatine kinase B and chloride intracellular channel protein 1. Functional classification of the proteins by database and the literature mining reveals their association with neuronal development and myelinogenesis, suggesting possible disease processes that mediate neurodegeneration.     

Quantitative mass spectrometry to investigate epidermal growth factor receptor phosphorylation dynamics

Identifying proteins of signaling networks has received much attention, because an array of biological processes are entirely dependent on protein cross-talk and protein-protein interactions. Protein posttranslational modifications (PTM) add an additional layer of complexity, resulting in complex signaling networks. Of particular interest to our working group are the signaling networks of epidermal growth factor (EGF) receptor, a transmembrane receptor tyrosine kinase involved in various cellular processes, including cell proliferation, differentiation, and survival. Ligand binding to the N-terminal residue of the extracellular domain of EGF receptor induces conformational changes, dimerization, and (auto)-phosphorylation of intracellular tyrosine residues. In addition, activated EGF receptor may positively affect survival pathways, and thus determines the pathways for tumor growth and progression. Notably, in many human malignancies exaggerated EGF receptor activities are commonly observed. An understanding of the mechanism that results in aberrant phosphorylation of EGF receptor tyrosine residues and derived signaling cascades is crucial for an understanding of molecular mechanisms in cancer development. Here, we summarize recent labeling methods and discuss the difficulties in quantitative MS-based phosphorylation assays to probe for receptor tyrosine kinase (RTK) activity. We also review recent advances in sample preparation to investigate membrane-bound RTKs, MS-based detection of phosphopeptides, and the diligent use of different quantitative methods for protein labeling.     

Phosphorylation Stoichiometries of Human Eukaryotic Initiation Factors

Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation.     

Catalytic constants enable the emergence of bistability in dual phosphorylation

Dual phosphorylation of proteins is a principal component of intracellular signalling. Bistability is considered an important property of such systems and its origin is not yet completely understood. Theoretical studies have established parameter values for multistationarity and bistability for many types of proteins. However, up to now no formal criterion linking multistationarity and bistability to the parameter values characterizing dual phosphorylation has been established. Deciding whether an unclassified protein has the capacity for bistability, therefore requires careful numerical studies. Here, we present two general algebraic conditions in the form of inequalities. The first employs the catalytic constants, and if satisfied guarantees multistationarity (and hence the potential for bistability). The second involves the catalytic and Michaelis constants, and if satisfied guarantees uniqueness of steady states (and hence absence of bistability). Our method also allows for the direct computation of the total concentration values such that multistationarity occurs. Applying our results yields insights into the emergence of bistability in the ERK–MEK–MKP system that previously required a delicate numerical effort. Our algebraic conditions present a practical way to determine the capacity for bistability and hence will be a useful tool for examining the origin of bistability in many models containing dual phosphorylation.     

Piperine Targets Different Drug Resistance Mechanisms in Human Ovarian Cancer Cell Lines Leading to Increased Sensitivity to Cytotoxic Drugs

Our goal was to examine the anticancer effects of piperine against the resistant human ovarian cancer cells and to explore the molecular mechanisms responsible for its anticancer effects. Our study used drug-sensitive ovarian cancer cell line W1 and its sublines resistant to paclitaxel (PAC) and topotecan (TOP). We analyzed the cytotoxic effect of piperine and cytostatic drugs using an MTT assay. The impact of piperine on protein expression was determined by immunofluorescence and Western blot. We also examined its effect on cell proliferation and migration. We noticed a different level of piperine resistance between cell lines. Piperine increases the cytotoxic effect of PAC and TOP in drug-resistant cells. We observed an increase in PTPRK expression correlated with decreased pTYR level after piperine treatment and downregulation of P-gp and BCRP expression. We also noted a decrease in COL3A1 and TGFBI expression in investigated cell lines and increased COL3A1 expression in media from W1PR2 cells. The expression of Ki67 protein and cell proliferation rate decreased after piperine treatment. Piperine markedly inhibited W1TR cell migration. Piperine can be considered a potential anticancer agent that can increase chemotherapy effectiveness in cancer patients.     

Regulatory role of phosphoproteins in the development of bovine small intestine during early life

The small intestine is the primary site of nutrient digestion and absorption, which plays a key role in the survival of neonatal calves. A comprehensive assessment of the phosphoproteomic changes in the small intestine of neonatal calves is unavailable; therefore, we used phosphopeptide enrichment coupled with liquid chromatography-tandem mass spectrometry to investigate the changes in the phosphoproteome profile in the bovine small intestine during the first 36 h of life. Twelve neonatal male calves were assigned to one of the following groups: (1) calves not fed colostrum and slaughtered approximately 2 h postpartum (n = 3), (2) calves fed colostrum at 1 to 2 h and slaughtered 8 h postpartum (n = 3), (3) calves fed 2 colostrum meals (at 1–2 and 10–12 h) and slaughtered 24 h postpartum (n = 3), (4) calves fed 3 colostrum meals (at 1–2, 10–12, and 22–24 h) and slaughtered 36 h postpartum (n = 3). Mid-duodenal, jejunal, and ileal samples of the calves were collected after slaughter. We identified 1,678 phosphoproteins with approximately 3,080 phosphosites, which were mainly Ser (89.9%), Thr (9.8%), and Tyr (0.3%) residues; they belonged to the prodirected (52.9%), basic (20.4%), acidic (16.6%), and Tyr-directed (1.7%) motif categories. The regional differentially expressed phosphoproteins included zonula occludens 2, sorting nexin 12, and protein kinase C, which are mainly associated with developmental processes, intracellular transport, vesicle-mediated transport, and immune system process. They are enriched in the endocytosis, tight junction, insulin signaling, and focal adhesion pathways. The temporal differentially expressed phosphoproteins included occludin, epsin 1, and bridging integrator 1, which were mainly associated with macromolecule metabolic process, cell adhesion, and growth. They were enriched in the spliceosomes, adherens junctions, and tight junctions. The observed changes in the phosphoproteins in the tissues of small intestine suggest the protein phosphorylation plays an important role in nutrient transport and immune response of calves during early life, which needs to be confirmed in a larger study.