Functional Activity Of Protein Research Articles

Белки, связанные с фактором Н, в патогенезе атипичного гемолитико-уремического синдрома

Atypical hemolytic-uremic syndrome (aHUS) is a rare disease of the group of thrombotic microangiopathies caused by hyperactivation of an alternative pathway of the complement system. One of the mechanisms of aHUS development is the change in the functional activity of proteins of the factor H (FH) family. We analyzed the available data on the influence of factor H protein family on aHUS development. A total of 43 studies published in 2003-2021, including 41 foreign ones, were reviewed. PubMed, Web of Science, Scopus, еLibrary, Google Scholar databases were searched using keywords: aHUS, factor H, factor H-like protein, factor H-related proteins (FHRs). Priority was given to the results of randomized controlled trials, systematic reviews with meta-analysis. Duplicate articles were excluded. Based on the analysis results, conclusions were drawn that disruption of C3b subunit inactivation as a result of changes in the functional activity of FH family proteins mediates the formation of a membrane-attacking complex. Among the mechanisms of FH, FHR1, and FHR3 inhibition are genetic aberrations (missense mutations, reading frame shift), formation of hybrid proteins, and synthesis of autologous antibodies. Other mechanisms of alternative pathway hyperactivation include decreased concentration or complete absence of FH, FHR1, FHR3, and FHR4 as a result of deletion of 1q31.3 fragments or formation of hybrid proteins. The change in the FH functional activity is also possible due to autoinactivation or competitive inhibition when the FHRs concentration increases.

Ganglioside GD3 regulates neural stem cell quiescence and controls postnatal neurogenesis.

The postnatal neural stem cell (NSC) pool hosts quiescent and activated radial glia-like NSCs contributing to neurogenesis throughout adulthood. However, the underlying regulatory mechanism during the transition from quiescent NSCs to activated NSCs in the postnatal NSC niche is not fully understood. Lipid metabolism and lipid composition play important roles in regulating NSC fate determination. Biological lipid membranes define the individual cellular shape and help maintain cellular organization and are highly heterogeneous in structure and there exist diverse microdomains (also known as lipid rafts), which are enriched with sugar molecules, such as glycosphingolipids. An often overlooked but key aspect is that the functional activities of proteins and genes are highly dependent on their molecular environments. We previously reported that ganglioside GD3 is the predominant species in NSCs and that the reduced postnatal NSC pools are observed in global GD3-synthase knockout (GD3S-KO) mouse brains. The specific roles of GD3 in determining the stage and cell-lineage determination of NSCs remain unclear, since global GD3S-KO mice cannot distinguish if GD3 regulates postnatal neurogenesis or developmental impacts. Here, we show that inducible GD3 deletion in postnatal radial glia-like NSCs promotes NSC activation, resulting in the loss of the long-term maintenance of the adult NSC pools. The reduced neurogenesis in the subventricular zone (SVZ) and the dentate gyrus (DG) of GD3S-conditional-knockout mice led to the impaired olfactory and memory functions. Thus, our results provide convincing evidence that postnatal GD3 maintains the quiescent state of radial glia-like NSCs in the adult NSC niche.

Isolation and identification of the new baicalin target protein to develop flavonoid structure-based therapeutic agents

Proteins are vital constituents of all living organisms. As many therapeutic agents alter the activity of functional proteins, identifying functional target proteins of small bioactive molecules isessential for the rational design of stronger medicines. Flavonoids with antioxidant, anti-allergy, and anti-inflammatory effects are expected to have preventive effects for several diseases closely related to oxidation and inflammation, including heart disease, cancer, neurodegenerative disorders, and eye diseases. Therefore, identifying the proteins involved in the pharmacological actions of flavonoids, and designing a flavonoid structure-based medicine that strongly and specifically inhibits flavonoid target proteins, could aid the development of more effective medicines for treating heart disease, cancer, neurodegenerative disorders, and ocular diseases with few side effects. To isolate the flavonoid target protein, we conducted a novel affinity chromatography in a column wherein baicalin, a representative flavonoid, was attached to Affi-Gel 102. Through affinity chromatography and nano LC-MS/MS, we identified GAPDH as a flavonoid target protein. Then, we performed fluorescence quenching and an enzyme inhibition assay to experimentally confirmbaicalin’s binding affinity for, and inhibition of, GAPDH. We also conducted in silico docking simulations to visualize the binding modes of baicalin and the newly identified flavonoid target protein, GAPDH. From the results of this study, it was considered that one of the reasons why baicalin exhibits the effects on cancer and neurodegenerative diseases is that it inhibits the activity of GAPDH. In summary, we showed that Affi-Gel102 could quickly and accurately isolate the target protein for bioactive small molecules, without the need for isotopic labeling or a fluorescent probe. By using the method presented here, it was possible to easily isolate the target protein of a medicine containing a carboxylic acid.

Effect of cholesterol on the ion-membrane interaction: Zeta potential and dynamic light scattering study

Cholesterol in a bio-membrane plays a significant role in many cellular event and is known to regulate the functional activity of protein and ion channel. In this study we report a significant effect of cholesterol on the ion-membrane interaction. We prepare large unilamellar vesicles, composed of zwitterionic lipid DOPC and anionic lipid DOPG with different cholesterol concentration. Electrostatics of anionic membranes containing cholesterol in the presence of NaCl has systematically been explored using dynamic light scattering and zeta potential. Negative zeta potential of the membrane decreases its negative value with increasing ion concentration for all cholesterol concentrations. However, zeta potential itself decreases with increasing cholesterol content even in the absence of monovalent ions. Electrostatic behaviour of the membrane is determined from well-known Gouy Chapmann model. Negative surface charge density of the membrane decreases with increasing cholesterol content. Binding constant, estimated from the electrostatic double layer theory, is found to increase significantly in the presence of cholesterol. Comparison of electrostatic parameters of the membrane in the presence and absence of cholesterol suggests that cholesterol significantly alter the electrostatic behaviour of the membrane.

Spy chemistry enables stable protein immobilization on iron oxide nanoparticles with enhanced magnetic properties

Iron oxide nanoparticles (IONPs) modified with functional proteins hold great promise in the biomedical field. However, conventional protein modification strategies, such as adsorption and covalent coupling, are either unstable or nonspecific, or may result in the changes of protein structure and ultimately the loss of protein activity. Modification of active proteins on small-sized IONPs with a particle size of less than 30 nm is especially difficult due to their high surface energy. Herein, we developed a universal modification method based on Spy chemistry for rapid and stable protein immobilization on small-sized IONPs, which only requires the presence of active groups on the surface of nanoparticles that can couple with SpyCatcher. In short, the SpyCatcher peptides were first coated on the surface of IONPs by cross-linking with activated groups, and then the SpyTag peptide fused with a model protein (enhanced green fluorescent protein, EGFP) was engineered (SpyTag-EGFP) and directly coupled to SpyCatcher-modified IONPs by self-assembly, which is spontaneous and robust while avoiding the effect of chemical reactions on functional protein activity. The obtained EGFP-functionalized IONPs exhibited enhanced and stable green fluorescence and improved magnetic properties. In addition, the cell internalization efficiency of EGFP-functionalized IONPs was significantly increased as compared to unmodified IONPs, providing an ideal solution for efficient cell labeling and tracking. In conclusion, here we report a rapid and easy strategy for EGFP immobilization on IONPs based on Spy chemistry, which could be further adapted to other functional proteins in the future. SpyCatcher-modified IONPs and SpyTag-X (arbitrary functional fusion proteins) hold great potential to be applied as a versatile platform for protein immobilization on IONPs and enable its multifunctional application in the future.

Insertion of exogenous genes within the ORF1b coding region of porcine astrovirus

Porcine astrovirus (PAstV) is a common cause of diarrhea in swine farms. The current understanding of the molecular virology and pathogenesis of PAstV is incomplete, especially due to the limited functional tools available. Here, ten sites in the open reading frame 1b (ORF1b) of the PAstV genome were determined to tolerate random 15 nt insertions based on the infectious full-length cDNA clones of PAstV using transposon-based insertion-mediated mutagenesis of three selected regions of the PAstV genome. Insertion of the commonly used Flag tag into seven of the ten insertion sites allowed the production of infectious viruses and allowed their recognition by specifically labeled monoclonal antibodies. Indirect immunofluorescence showed that the Flag-tagged ORF1b protein partially overlapped with the coat protein within the cytoplasm. An improved light-oxygen-voltage (iLOV) gene was also introduced into these seven sites, and only one viable recombinant virus that expressed the iLOV reporter gene at the B2 site was recovered. Biological analysis of the reporter viruses showed that these exhibited similar growth characteristics to the parental virus, but they produced fewer infectious virus particles and replicated at a slower rate. The recombinant viruses containing iLOV fused to ORF1b protein, which maintained their stability and displayed green fluorescence for up to three generations after passaging in cell culture. The porcine astroviruses (PAstVs) expressing iLOV were then used to assess the in vitro antiviral activities of mefloquine hydrochloride and ribavirin. Altogether, the recombinant PAstVs expressing iLOV can be used as a reporter virus tool for the screening of anti-PAstV drugs as well as the investigation of PAstV replication and the functional activities of proteins in living cells.

Probing the antioxidant activity of functional proteins and bioactive peptides in Hermetia illucens larvae fed with food wastes

Food waste is becoming more prevalent, and managing it is one of the most important issues in terms of food safety. In this study, functional proteins and bioactive peptides produced from the enzymatic digestion of black soldier fly (Hermetia illucens L., BSF) fed with food wastes were characterized and quantified using proteomics-based analysis. The results revealed approximately 78 peptides and 57 proteins, including 40S ribosomal protein S4, 60S ribosomal protein L8, ATP synthase subunit alpha, ribosomal protein S3, Histone H2A, NADP-glutamate dehydrogenase, Fumarate hydratase, RNA helicase, Chitin binding Peritrophin-A, Lectin C-type protein, etc. were found in BSF. Furthermore, functional analysis of the proteins revealed that the 60S ribosomal protein L5 (RpL5) in BSF interacted with a variety of ribosomal proteins and played a key role in the glycolytic process (AT14039p). Higher antioxidant activity was found in peptide sequences such as GYGFGGGAGCLSMDTGAHLNR, VVPSANRAMVGIVAGGGRIDKPILK, AGLQFPVGR, GFKDQIQDVFK, and GFKDQIQDVFK. It was concluded that the bioconversion of food wastes by BSF brought about the generation of a variety of functional proteins and bioactive peptides with strong antioxidant activity. However, more studies are required to exploit BSF's potential in the value addition of food wastes.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

МОЛЕКУЛЯРНО-ГЕНЕТИЧЕСКИЕ МЕХАНИЗМЫ УСТОЙЧИВОСТИ НОЗОКОМИАЛЬНЫХ ШТАММОВ KLEBSIELLA PNEUMONIAE К ПОЛИМИКСИНАМ И АНТИБИОТИКАМ ДРУГИХ ГРУПП ПО ДАННЫМ ПОЛНОГЕНОМНОГО СЕКВЕНИРОВАНИЯ

Identification of numerous mechanisms of resistance to colistin and other antibiotics is possible using whole genome sequencing. Objectives. To assess the molecular-genetic mechanisms of resistance to polymyxins and antibiotics of other groups in nosocomial Klebsiella pneumoniae strains. Material and methods. For 13 multidrug- and extensively drug-resistant K.pneumoniae strains semiconductor sequencing was performed in the Ion PGM System genomic sequencer (Thermo Fisher Scientific, USA). The assembly of genomic sequences and their annotation were carried out. The PROVEAN software tool was used to predict the influence of nucleotide replacements on the structure of amino acid sequences and functional activity of proteins. The identification of antibiotic resistance genes and the search for efflux mechanisms were performed by the ResFinder v.4.1 and CARD web resources. Results. Several types of β-lactamase genes were detected simultaneously in all strains, as well as genes of resistance to fosfomycin. Genes of resistance to aminoglycosides were identified in 11 strains, to chloramphenicol - in 10, to rifampicin - in 5, to macrolides - in 4. The mcr phosphoethanolamine transferase genes were absent in all strains. Functionally significant substitutions were revealed in the pmrB gene (D150Y, T157P, G207S) comparing the studied samples with the reference K. pneumoniae strain ATCC 700603. Changes in the mgrB gene were also found in colistin-resistant strains (W20R replacement, insertional inactivation of the gene by transposons of the IS1, IS4 and IS5 families). Conclusions. The results of whole genome sequencing represent the significant resistance of nosocomial Klebsiella pneumoniae strains to the majority of antibiotics including β-lactams, aminoglycosides, fluoroquinolones, fosfomycin, chloramphenicol, polymyxins. Genetic determinants of colistin resistance were revealed (insertional inactivation and deletion of the mgrB gene; D150Y, T157P and G207S substitutions in the pmrB gene) in strains with colistin MIC 64-128 mg/l and their absence in colistin-susceptible strains.

Strontium Binding to α-Parvalbumin, a Canonical Calcium-Binding Protein of the “EF-Hand” Family

Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr2+ interference with Ca2+ binding to proteins of the EF-hand family, we studied Sr2+/Ca2+ interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca2+ binding sites of recombinant human α-PA (‘CD’ and ‘EF’ sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 109 M−1 and 4 × 109 M−1 for Ca2+, and 2 × 107 M−1 and 2 × 106 M−1 for Sr2+. Inactivation of the EF site by homologous substitution of the Ca2+-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca2+/Sr2+ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca2+/Sr2+ affinity. These results suggest that Sr2+ and Ca2+ bind to CD/EF sites of α-PA and the Ca2+/Sr2+ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr2+ titration of the Ca2+-loaded α-PA revealed presence of secondary Sr2+ binding site(s) with an apparent equilibrium association constant of 4 × 105 M−1. Fourier-transform infrared spectroscopy data evidence that Ca2+/Sr2+-loaded forms of α-PA exhibit similar states of their COO− groups. Near-UV circular dichroism (CD) data show that Ca2+/Sr2+ binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca2+/Sr2+ binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr2+-induced increase in stability of tertiary structure of α-PA, compared to the Ca2+-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca2+-binding sites of α-PA are well protected against exchange of Ca2+ for Sr2+ regardless of coordination number of Sr2+, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca2+/Sr2+ selectivity. Overall, despite lowered affinity of α-PA to Sr2+, the latter competes with Ca2+ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr2+ binding site(s) could be a factor contributing to Sr2+ impact on the functional activity of proteins.

Glutathione in Protein Redox Modulation through S-Glutathionylation and S-Nitrosylation.

S-glutathionylation and S-nitrosylation are reversible post-translational modifications on the cysteine thiol groups of proteins, which occur in cells under physiological conditions and oxidative/nitrosative stress both spontaneously and enzymatically. They are important for the regulation of the functional activity of proteins and intracellular processes. Connecting link and “switch” functions between S-glutathionylation and S-nitrosylation may be performed by GSNO, the generation of which depends on the GSH content, the GSH/GSSG ratio, and the cellular redox state. An important role in the regulation of these processes is played by Trx family enzymes (Trx, Grx, PDI), the activity of which is determined by the cellular redox status and depends on the GSH/GSSG ratio. In this review, we analyze data concerning the role of GSH/GSSG in the modulation of S-glutathionylation and S-nitrosylation and their relationship for the maintenance of cell viability.

Interactions of dendrimers and dendronized nanoparticles with proteins

Dendrimers are hyperbranched polymers belonging to the class of nanomaterials. These nanostructures and their derivatives (dendrons and dendronized nanoparticles) are multi-target nanocarriers that can be modified to achieve various tasks. For example, it is possible to increase biocompatibility and stability, to control the release of active substances. Their widespread use in biology and medicine requires an understanding of the basic mechanisms of their interaction with proteins - one of the main biological systems. The interaction of dendrimers with proteins may vary depending on the size of the ones, surface charge, structure and stiffness of the branches. Here, both electrostatic interactions arising due to the presence of dendrimers and surface charge proteins, as well as hydrophobic ones, due to the corresponding cavities in the structure of dendrimers, can manifest themselves here. The formation of hydrogen bonds is possible. All these interactions in one way or another can affect the structure and functions of proteins. Present article discusses the mechanisms of interactions between dendrimers, dendronized nanoparticles and protein macromolecules. The effect of nanoparticles on the secondary structure, conformation, dynamics and functional activity of proteins is reviewed. The different models for dendrimer-protein interactions are described.

Abstract 4384: Quantitative assessment of drug combination effects in NSCLC cell lines through network-based analysis of functional protein activity

Abstract Non-small cell lung cancer (NSCLC) is the most prevalent type of lung cancer, which is the leading cause of cancer death worldwide. The KRAS oncogene is one of the most common driver mutations in NSCLC patients but, despite that, there are no approved targeted therapies for tumors harboring this mutation. The MEK inhibitor Selumetinib (SE) turned out to be an ineffective drug against KRAS mutated lung cancer. Since combination therapy has shown to be beneficial in many tumors, we wanted to investigate the combination of MEK inhibition with other targeted therapies. Thus, we measured the proteomic response of KRAS mutant (MUT) and wild-type (WT) NSCLC cell lines (CLs) to the combination of SE with Everolimus (EV), an mTOR inhibitor, and Tamoxifen (TA), an Estrogen inhibitor. The expression level of 183 proteins was measured through Reverse Phase Protein Array (RPPA) in eight NSCLC CLs treated with SE, SE plus EV and SE plus TA at 6 time points: 5 minutes (min), 30 min, 1, 2, 6 and 24 hours (h). As control, measures were taken in baseline CLs and in CLs with only dimethyl sulfoxide. To study the drug combination effects, we applied a computational workflow centered on two algorithms initially developed for gene expression data. We analyzed the CLs with the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) to reconstruct a context specific signaling network of 2635 interactions between 73 kinases and 6 phosphatases and their substrates. Then, we analyzed the obtained network with the Virtual Inference of Protein activity by Enriched Regulon analysis (VIPER) algorithm to assess the mechanism of action of the drug compounds and how they dysregulate the activity of kinases and phosphatases over time. With respect to SE alone and apart from the drug targets, VIPER predicted the following proteins as highly inhibited over time across all CLs : 1) Aurora and LKB1 in SE+EV, 2) AMPK, c-RAF and ATR in SE+TA, 3) mTOR, p90, p70S6K, c-MET and GSK-3 in both combinations. On the other hand, Akt, ETK, Axl and FAK are all activated in SE+EV as well as PP2A, SGK1 and IGF1R in SE+TA, after 1h from the treatment. By running Gene Set Enrichment Analysis on the VIPER output against the primary drug target pathways, we found that mTOR signaling pathway exhibits a quite different evolution between KRAS MUT and WT CLs. On the other hand, other pathways revealed mechanisms of response that are CL specific. For instance, the main MEK pathways in H1734 CL (KRAS MUT) treated with SE+TA are all insensitive to the drug compounds. In conclusion, this computational approach successfully predicted protein-protein interactions and elucidated both proteomic mechanisms of drug combinations and CL specific dependencies. It allows to develop effective predictive and quantitative models reproducing each CL behavior, in order to realize the fullest potential of a targeted therapeutic approach. Citation Format: Chiara Antonini, George Rosenberger, Fortunato Bianconi, Lorenzo Tomassoni, Vienna Ludovini, Sara Baglivo, Sara Calandrini, Elisa Baldelli, Mariaelena Pierobon, Emanuel F. Petricoin, Andrea Califano. Quantitative assessment of drug combination effects in NSCLC cell lines through network-based analysis of functional protein activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4384.

Physicochemical properties and bioactivity of whey protein isolate-inulin conjugates obtained by Maillard reaction

The functional properties and physiological functions of whey protein isolate (WPI) decreased near its isoelectric point (PI). The Maillard reaction covalently binding polysaccharides to proteins is an effective method to improve the functional activities of proteins. WPI-inulin conjugates were prepared by wet-heating method at 70 °C for 2 h, 4 h and 6 h, respectively. New bonds at higher molecular zone appearing at SDS-PAGE, decreased free amino acid content and new formed CN bonds in FT-IR of conjugates compared with WPI confirmed the formation of the covalent bonds between WPI and inulin. As the increase of the reaction time, both the brown intensity and fluorescence intensity of WPI-inulin conjugates became higher. Amino acid contents, Circular dichroism analysis and SEM analysis presented the primary structure, secondary structure and surface structure change of protein after covalent with inulin. Emulsion properties of emulsion activity (EAI) and emulsion stability (ES) of WPI-inulin conjugates were assessed and both showed significantly enhanced compared with WPI at range of pH 3 to pH 7. AAPH+ scavenging test and ORAC measurement also revealed that covalent binding with inulin enhanced the antioxidant activities of WPI. This work presented the conjugation with inulin successfully enhanced the functional properties of WPI.

Blueberry VcLon1 protease increases iron use efficiency by alleviating chloroplast oxidative stress

Aim to unveil the functions of VcLon1 in plant Fe use efficiency. RT-PCR was used to analyze the expression profile of VcLon1. VcLon1 was expressed in Nicotiana benthamiana, and its homologous tobacco gene was silenced using RNAi. The differences in biomass growth, oxidative stress and chloroplast ultrastructure between wild type and transgenic Nicotiana benthaminana were analyzed after being subjected to Fe deficiency stress. The RT-PCR showed that the expression of VcLon1 was significantly higher in young leaves than in old leaves or in leaves treated with Fe deficiency stress, indicating that VcLon1 is involved in reactive oxygen species (ROS) homeostasis induced by senescence or Fe deficiency. Compared with wild-type, overexpression of VcLon1 in Nicotiana benthamiana reduced damage to chloroplast structure induced by Fe deficiency. Furthermore, the contents of H2O2, MDA and carbonylated protein in leaves were kept at a low level, and antioxidant enzyme activities in chloroplasts such as SOD and APX are also generally higher in chloroplasts with VcLon1 overexpression. In contrast, the oxidative stress levels in NbLon1 RNAi silenced Nicotiana benthamiana leaves showed opposite trends. Under Fe deficiency stress, VcLon1 reduces oxidative damage in plants by degrading carbonylated proteins in organelles such as chloroplasts and effectively maintains the structure and function of organelles and the activity of functional proteins, contributing to Fe use efficiency in plants.

Detection of Posttranslational Modifications by Fluorescent Staining of Two-Dimensional Gels.

Posttranslational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial for the discovery of disease biomarkers. Fluorescent dyes specifically staining protein PTMs such as phosphorylation and glycosylation enable the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular interactions of many proteins are determined by their extent of phosphorylation. In our search for biomarkers of neurodegenerative diseases such as multiple sclerosis (MS), using an animal model, experimental autoimmune encephalomyelitis (EAE), we have applied the phosphorylation-specific fluorescent dye, ProQ Diamond, to study changes taking place in the phosphoproteome. Subsequent colloidal Coomassie staining of the same gels detects the changes at the whole proteome level. We have detected many changes taking place in the CNS tissue of the EAE animals at the whole proteome as well as at the phosphoproteome level resulting in valuable insights into the pathophysiological mechanism of EAE and MS.

The ribosome in action: Tuning of translational efficiency and protein folding.

The cellular proteome is shaped by the combined activities of the gene expression and quality control machineries. While transcription plays an undoubtedly important role, in recent years also translation emerged as a key step that defines the composition and quality of the proteome and the functional activity of proteins in the cell. Among the different post-transcriptional control mechanisms, translation initiation and elongation provide multiple checkpoints that can affect translational efficiency. A multitude of specific signals in mRNAs can determine the frequency of translation initiation, choice of the open reading frame, global and local elongation velocities, and the folding of the emerging protein. In addition to specific signatures in the mRNAs, also variations in the global pools of translation components, including ribosomes, tRNAs, mRNAs, and translation factors can alter translational efficiencies. The cellular outcomes of phenomena such as mRNA codon bias are sometimes difficult to understand due to the staggering complexity of covariates that affect codon usage, translation, and protein folding. Here we summarize the experimental evidence on how the ribosome-together with the other components of the translational machinery-can alter translational efficiencies of mRNA at the initiation and elongation stages and how translation velocity affects protein folding. We seek to explain these findings in the context of mechanistic work on the ribosome. The results argue in favour of a new understanding of translation control as a hub that links mRNA homeostasis to production and quality control of proteins in the cell.

Phosphorylation and Degradation of Tomosyn-2 De-represses Insulin Secretion

The abundance and functional activity of proteins involved in the formation of the SNARE complex are tightly regulated for efficient exocytosis. Tomosyn proteins are negative regulators of exocytosis. Tomosyn causes an attenuation of insulin secretion by limiting the formation of the SNARE complex. We hypothesized that glucose-dependent stimulation of insulin secretion from β-cells must involve reversing the inhibitory action of tomosyn. Here, we show that glucose increases tomosyn protein turnover. Within 1 h of exposure to 15 mM glucose, ~50% of tomosyn was degraded. The degradation of tomosyn in response to high glucose was blocked by inhibitors of the proteasomal pathway. Using (32)P labeling and mass spectrometry, we showed that tomosyn-2 is phosphorylated in response to high glucose, phorbol esters, and analogs of cAMP, all key insulin secretagogues. We identified 11 phosphorylation sites in tomosyn-2. Site-directed mutagenesis was used to generate phosphomimetic (Ser → Asp) and loss-of-function (Ser → Ala) mutants. The Ser → Asp mutant had enhanced protein turnover compared with the Ser → Ala mutant and wild type tomosyn-2. Additionally, the Ser → Asp tomosyn-2 mutant was ineffective at inhibiting insulin secretion. Using a proteomic screen for tomosyn-2-binding proteins, we identified Hrd-1, an E3-ubiquitin ligase. We showed that tomosyn-2 ubiquitination is increased by Hrd-1, and knockdown of Hrd-1 by short hairpin RNA resulted in increased abundance in tomosyn-2 protein levels. Taken together, our results reveal a mechanism by which enhanced phosphorylation of a negative regulator of secretion, tomosyn-2, in response to insulin secretagogues targets it to degradation by the Hrd-1 E3-ubiquitin ligase.

Inherited Thrombophilia and Recurrent Pregnancy Loss

Background:Recurrent pregnancy loss (RPL) is a common health problem. The polymorphisms G20210A of prothrombin gene (FII G 20210A), and G 1691A of factor V gene (Factor V Leiden, FVL) are the most extensively studied thrombophilic mutations in association to recurrent miscarriage.Objectives:To determine the frequency of FII G20210A and FVL polymorphisms as well as protein C and protein S deficiency in a series of patients with RPL compared with control group.Patients and Methods:The study group included 90 randomly selected patients with three or more consecutive miscarriages with the same partner in <20 weeks gestation in 2012. The control population consisted of 44 age-matched women with at least one live born children and no history of pregnancy loss. Functional activity of protein C and S, activated protein C resistance, FVL assay by polymerase chain reaction and prothrombin gene mutation were assessed. The polymorphism frequencies were recorded for each group and comparisons were made.Results:The mean functional activity of protein C and protein S were not significantly different between case and control groups (P >0.05). Frequency of protein C deficiency was also not significantly different between the case and control groups (P=0.906), but frequency of protein S deficiency was significantly higher in patients than controls (P=0.03). Genotype pattern of the patients and healthy individuals were not significantly different with regard to either FVL or Prothrombin G20210A (P > 0.05).Conclusions:We determined a significant higher frequency of protein S deficiency in patients with RPL compared with controls. But the frequency of protein C deficiency and the frequency of two common thrombophilic mutations (Factor V Leiden and Prothrombin G20210A), were not significantly different between patients with recurrent miscarriage and healthy women.

Protein Engineering For Directed Immobilization

Much effort has been put into the optimization of the functional activity of proteins. For biosensors this protein functional optimization will increase the biosensor's sensitivity and/or selectivity. However, the strategy chosen for the immobilization of the proteins to the sensor surface might be equally important for the development of sensor surfaces that are optimally biologically active. Several studies published in recent years show that the oriented immobilization of the bioactive molecules improves the sensor's properties. In this review, we discuss the state of the art of the different protein immobilization strategies that are commonly used today with a special focus on biosensor applications. These strategies include nonspecific immobilization techniques either by physical adsorption, by covalent coupling, or by specific immobilization via site-specifically introduced tags or bio-orthogonal chemistry. The different tags and bio-orthogonal chemistry available and the techniques to site-specifically introduce these groups in proteins are also discussed.