Huntington disease is a neurodegenerative disease characterized by a polymorphic tract of polyglutamine repeats in exon 1 of the huntingtin protein, which is thought to be responsible for protein aggregation and neuronal death. The polyglutamine tract is preceded by a 17-residue sequence that is intrinsically disordered. This region is subject to phosphorylation, acetylation and other post-translational modifications in vivo, which modulate its secondary structure, aggregation and, subcellular localization. We used Molecular Dynamics simulations with a novel Hamiltonian-replica-exchange-based enhanced sampling method, SWISH, and an optimal combination of water and protein force fields to study the effects of phosphorylation and acetylation as well as cross-talk between these modifications on the huntingtin N-terminus. The simulations, validated by circular dichroism, were used to formulate a mechanism by which the modifications influence helical conformations. Our findings have implications for
Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukar
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The current release not only provides the sequence-based information, but also annotates the structure-based information for protein post-translational modification. The interface is also designed to facilitate the access to the resource. This effective database is now freely accessible at http://db …
The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N-termini are hotspots of conserved post-translational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Our lab has identified novel post-translational modifications on human CENP-A N-termini using high-resolution MS. These include the trimethylation of Gly1 at the alpha-amino position and side-chain phosphorylation of Ser16 and Ser18. CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We identified the methyltransferase NRMT as the enzyme responsible for modifying the CENP-A amino terminus. Methylation occurs in the pre-nucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in pre-nucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal ...
Histones and their variants are subjected to several post-translational modifications (PTMs). Histones PTMs play an important role in the regulation of gene expression and are critical for the development and progression of many types of cancer, including breast cancer. In this study, we used two-dimensional TAU/SDS electrophoresis, coupled with mass spectrometry for a comprehensive profiling of histone PTMs in breast cancer cell lines.Proteomic approach allowed us to identify 85 histone PTMs, seventeen of which are not reported in the UniProt database. Western blot analysis was performed to confirm a peculiar pattern of PTMs in the sporadic and hereditary breast cancer cell lines compared to normal cells. Overlapping mass spectrometry data with western blotting results, we identified, for the first time to our knowledge, a tyrosine phosphorylation on histone H1, which is significantly higher in breast cancer cells. Additionally, by inhibiting specific signaling paths, such as PI3K, PPARγ and FAK
Post-translational protein processing refers to the folding, sorting, cleavage, and modifications required to make a protein functional after it is translated.
Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in...
In animals NO has been shown to play a key role in many important physiological process such as relaxation of vascular smooth muscle, neurotransmission, inflammation and immune function (Ignarro & Buga, 1987; ODell et al., 1991; Eiserich et al., 1998). Similarly, NO signalling in plants modulates a variety of physiological systems, from adaptive responses to germination, root growth and dynamics of stomatal aperture control. A significant emerging theme is the regulation of these processes through post-translational protein modifications, mainly metal nitrosylation, S-nitrosylation and tyrosine nitration (Besson-Bard et al., 2008). Metal nitrosylation is characterized by the formation of a NO-metal-containing protein and is best exemplified by the reaction between NO and haemoglobin (Hb), which controls vascular oxygen distribution. Metal nitrosylation also has a well characterized role during the activation of soluble guanylate cyclase (sGC) in animal cells, a classical route for the transfer ...
Asparagine-linked glycosylation is one of the most common protein modification reactions in eukaryotic cells, occurring on N-(x≠P)-T/S/C consensus sequons on newly synthesized proteins in the lumen of the rough endoplasmic reticulum (RER). Transfer of the preassembled oligosaccharide (GlcNAc2Man9Glc3) from a dolichol pyrophosphate carrier to sequons is mediated by the oligosaccharyltransferase (OST). Although N-glycosylation is often described as a post-translational protein modification reaction, most N-linked glycans are added to ribosome-bound nascent polypeptides as the protein is passing through the protein translocation channel into the lumen of the RER. A cotranslational mode of N-linked glycosylation ensures that addition of glycan occurs before protein folding, thereby relieving the restriction that sequons reside on surface-exposed loops or within disordered protein segments that can access the OST active site (Lizak et al., 2011).. The OST is a hetero-oligomeric integral membrane ...
Molecular Reproduction and Development takes an integrated, systems-biology approach to understand the dynamic continuum of cellular, reproductive, and developmental processes. This journal fosters dialogue among diverse disciplines through primary research communications and educational forums, with the philosophy that fundamental findings within the life sciences result from a convergence of disciplines.. Increasingly, readers of the Journal need to be informed of diverse, yet integrated, topics impinging on their areas of interest. This requires an expansion in thinking towards non-traditional, interdisciplinary experimental design and data analysis. For example, biologists need to know how nanodevices might be used, while bioengineers need to know how post-translational protein modifications affect developmental mechanisms. The Journal will provide a means for readers to integrate divergent scientific disciplines into their current and future research. Readers will turn to Molecular ...
Demonstrates the first synthesis of histones, nucleosome core particles and defined nucleosomes arrays bearing this important post-translational modification, and demonstrates using single molecule FRET that H3K56 acetylation increases DNA breathing on nucleosomes. This paper demonstrates the power of creating designer nucleosomes to address problems in chromatin biology. More broadly, it demonstrates the power of genetically installing post translational modifications for asking previously un-addressable questions about biological regulation. ...
Nitric oxide (NO) is a free-radical product of mammalian cell metabolism that plays diverse and important roles in the regulation of cell function. Biological actions of NO arise as a direct consequence of chemical reactions between NO or NO-derived species and protein targets. Reactions of NO with transition metals in target proteins have garnered the most attention to date as the principal mechanism of NO signaling; nonetheless, S-nitrosylation of protein Cys residues is rapidly moving to center stage in importance. In general, however, there has been a delay in adequate appreciation of the role of S-nitrosylation in biological signaling by NO. This lag is attributed to a poor understanding of the basis for selective targeting of NO to particular thiols, and methodological limitations in accurately quantifying this modification--recent breakthroughs in concepts and methods diminish these barriers. Here, we consider the wheres and whys of protein S-nitrosylation and its basis for specificity. ...
Phosphorylation is a crucial post-translational protein modification mechanism with important regulatory functions in biological systems. It is catalyzed by a group of enzymes called kinases, each of which recognizes certain target sites in its substrate proteins.
The Proteomics Core Facility at the Frankfurt location is an initiative of the DKTK and is equipped with three Q Exactive mass spectrometers from Thermo Fisher (one Q Exactive, one Q Exactive Plus and one Q Exactive HF instrument). The available technology can be used to e.g. identify and quantify protein expression patterns, post-translational protein modifications such as phosphorylation, acetylation and ubiquitination, and protein complexes.. This makes it possible to conduct a comprehensive characterisation of oncogenic mechanisms at the protein level, which includes mapping of oncogenic signal transduction processes. The technical advances made in recent years mean that it is now possible to identify and quantify in relative terms several thousand proteins from just a few micrograms of protein. The main activities of the Proteomics Core Facility relate to translational research topics in the field of acute leukaemia, lymphoma, lung cancer, rectal cancer and brain tumours. The research uses ...
Background endoplasmic reticulum lumen, arylsulfatase activity, glycosphingolipid metabolic process, post-translational protein modification Description ARSH Polyclonal Antibody, FITC Conjugated. FITC. Raised in Rabbit....
Medical & Life Sciences (Biochemistry), Manipulation and analysis of proteins; Research areas include the experimental analysis of enzyme mechanisms, post-translational protein modifications, proteomics, and protein-nucleic acid interactions studied in the biological context of cell cycle control, chromatin regulation and renewable energy research ...
The transfer of macromolecules such as nucleic acids and proteins to solid-phase membranous support is known as blotting. Fragments of DNA and RNA molecules separated by gel electrophoresis are transferred to a nylon or nitrocellulose membrane in a process termed as Southern and Northern blotting, respectively. Southern blotting was introduced by Edwin Southern in 1975 as a method to detect specific sequences of DNA in DNA samples. The other blotting techniques emerged from this method have been termed as Northern (for RNA), Western (for proteins), Eastern (for post-translational protein modifications) and Southwestern (for DNA-protein interactions) blotting.. Southern and Northern blotting protocols involve the following major steps:. ...
Post-Translational Modification - Most proteins undergo some form of modification following translation. These modifications result in mass changes that are detected during analysis. Post-translational modifications such as glycosylation, phosphorylation, and sulfation, to name a few, serve many functions. As a result, the analysis of proteins and their post-translational modifications is particularly important for the study of diseases where multiple genes are known to be involved, such as heart disease, cancer and diabetes.
With the rapidly increasing use of proteins as biotherapeutics to treat diseases, the characterization of these large molecules using mass spectrometry has become a highly attractive field of research. A particular area of research is the identification and characterization of protein post-translational modifications. Disulfide bonds and glycosylation are among the most critical protein post-translational modifications (PTMs), as they play vital roles in maintaining the proper protein folding, structure, and functions. These two PTMs are particularly important in the development and characterization of monoclonal antibody-based drugs, which are the most prevalent protein therapeutics in the market. Among the four classes of immunoglobulins (IgGs), the disulfide connectivity of IgG1, IgG2 and IgG4 have been effectively studied, and IgG2 and IgG4 have been shown to have disulfide bond-mediated isomers due to alternative disulfide bond connectivity. However, no studies to investigate the presence ...
Ngn3 is recognized as a regulator of pancreatic endocrine formation, and Notch signaling as an important negative regulator Ngn3 gene expression. By conditionally controlling expression of Ngn3 in the pancreas, we find that these two signaling components are dynamically linked. This connection involves transcriptional repression as previously shown, but also incorporates a novel post-translational mechanism. In addition to its ability to promote endocrine fate, we provide evidence of a competing ability of Ngn3 in the patterning of multipotent progenitor cells in turn controlling the formation of ducts. On one hand, Ngn3 cell-intrinsically activates endocrine target genes; on the other, Ngn3 cell-extrinsically promotes lateral signaling via the Dll1,Notch,Hes1 pathway which substantially limits its ability to sustain endocrine formation. Prior to endocrine commitment, the Ngn3-mediated activation of the Notch,Hes1 pathway impacts formation of the trunk domain in the pancreas causing multipotent ...
Inheritance of biological information to future generations depends on the replication of DNA and the Mendelian principle of distribution of genes. In addition, external and environmental factors can influence traits that can be propagated to offspring, but the molecular details of this are only beginning to be understood. The discoveries of DNA methylation and post-translational modifications on chromatin and histones provided entry points for regulating gene expression, an area now defined as epigenetics and epigenomics. Mass spectrometry turned out to be instrumental in uncovering molecular details involved in these processes. The central role of histone post-translational modifications in epigenetics related biological processes has revitalized mass spectrometry based investigations. In this special report, current approaches and future challenges that lay ahead due to the enormous complexity are discussed.
Protein glycation is a spontaneous PTM of the proteome focused mainly on N-terminal and lysine side chain amino groups by glucose forming fructosa- mine residues and on guanidino groups of arginine residues forming mainly MG-derived hydroimidazolone residues. The latter appears to be most functionally damaging in physiological systems. Glycation is a relatively labile PTM, and so customized preanalytical processing is required to avoid compromising mass spectrometric analysis outcomes. ETD fragmentation has a clear advantage for the analysis of the fructosamine proteome, whereas CID, HCD, and ETD may be used for arginine-based dihydroxyimidazolidine/hydroimi- dazolone detection. Robust procedures are available for the detection and quantitation of total glycation adducts in protein extracts, and these may be combined with proteomics analysis for added security of findings - particularly as it is still challenging to achieve high sequence coverage in proteomics experiments. LC-MS/MS quantitative ...
Step change for this strategy was achieved by introduction of a dimethylation step posttryptic digest to block free (N-terminal, lysine-e) amines thus increasing the difference in basicity between N-acetylated and the rest of peptides in the sample. One-step purification of N-acetylated peptides is achieved by solid-phase extraction, using SCX in batch mode. A key benefit of this approach is that it can be utilized with stable isotope-labeled dimethylation reagents for relative quantification, an approach which has found application to distinguish protein isoforms that are N-terminally acetylated but differ in N-terminal amino acid sequence, for example, p-actin/y-actin isoforms [111]. ...
Enrichment methods to facilitate detection and quantitation of PTM proteins are a common strategy in proteomics. A boronate affinity chromatography method has been used for the fructosamine proteome based on the binding of the cis-1,2-diol structure of fructosamine-modified proteins, with subsequent release from the boronate affinity matrix with weak acid. Although some enzymatically glycosylated proteins contain cis-1,2-diol moieties, steric effects, proximate negatively charged groups, and acetylation limit the retention and interference in this method by glycoproteins [98]. A similar affinity method is used in the routine separation of hemoglobin in clinical chemistry to quantify glycated hemoglobin HbA1c for the assessment of glycemic control in diabetes [99]. Boronate affinity enrichment of protein glycated by glucose was employed in a study of glycated proteins in human plasma and red blood cells, and 7749 unique glycated peptides corresponding to 3742 unique glycated proteins were ...
The aforementioned considerations are features relating to the rate of formation of glycation adducts. FL and MG-H1 residues have half-lives of ca. 25 and 12 days, respectively [19, 106], which exceed the half-lives of most human proteins (median half-life of 1.9 days [107]). Therefore, for many proteins, the steady-state extent of protein glycation is also influenced by the half-life of the protein. Hence, early studies found that the extent of glycation by glucose of several proteins in vivo was linked to the protein half-life [108]. Since glycation leads to protein distortion and misfolding, it is also expected that glycated proteins are targeted for cellular proteolysis and have an unusually decreased halflife. This remains to be determined in robust unfocused proteome dynamics studies. The level of FL and N-terminal fructosamine residues in cellular proteins is also influenced by enzymatic removal and repair by F3PK [109]. F3PK has different specific activity for FL residues in different ...
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E-cadherin is synthesized as a precursor and then undergoes cleavage by proprotein convertases. This processing is essential for E-cadherin maturation and cell adhesion. Loss of cell adhesion causes detachment-induced apoptosis- anoikis. Anoikis can be inhibited despite loss of cell-matrix interactions by preserving E-cadherin mediated cell-cell adhesion. Conversely, acute loss of E-cadherin sensitizes cells to apoptosis by unknown post-translational mechanisms. In response to drug treatment of breast cancer cells, our analysis revealed that two independent modifications of E-cadherin inhibit its cell surface transport. Firstly, O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of the cytoplasmic domain retains E-cadherin in the endoplasmic reticulum. Secondly, incomplete processing by proprotein convertases arrests E-cadherin transport late in the secretory pathway. We demonstrated these E-cadherin modifications (detected by specific lectins and antibodies) do not affect binding to ...
Large‐scale identification of PTMs across multiple species has opened the door to comparative studies regarding the evolutionary conservation of the modification sites, interactions and function. These studies have primarily focused on protein phosphorylation given its broad functional role and well established detection methods. Most of the comparative analyses performed to date have studied the conservation of the modified residues in alignments of orthologous proteins (Holt et al, 2009; Landry et al, 2009; Nguyen Ba & Moses, 2010; Gray & Kumar, 2011). There is some debate regarding the extent of conservation: some studies report little to no conservation of the modified residues when compared to unmodified amino‐acids (Holt et al, 2009; Landry et al, 2009; Nguyen Ba & Moses, 2010), whereas others observe a significant constraint due to the phosphorylation (Gray & Kumar, 2011). Overall, a reconciled view on these finding is that phosphorylated residues show a significant but small increase ...
Imagine you are doing XIC based label free quan on sample 1 and sample 2. In sample 1 you find peptide HAPPYK with an intensity of 1e6 that elutes at 23.4 minutes. In sample B, you only find HAPPYK at that retention time at almost baseline, but at 10.8 minutes you find HAPPphospho-YK with an intesity of 8e5. Unless youve got some awesome tools that I dont have for global analysis of this type, get ready to buckle down for a fun filled afternoon of manually linking your modified and unmodified variants in that Excel sheet! (PD 2.0/2.1 can make the job a little easier for you -- see video 22 here, but you still have to do a lot of manual work ...
ziyuBio offers hundreds of peptide modifications to meet your custom peptide needs. These peptide modifications can be used to create synthetic peptide with the exact conformation or characteristics needed for specific applications. Large numbers of modified amino acids are focus on post-translation modification (PTM) that naturally occur in vivo, while others are pharmacologically modified or stable isotope labeled. Additionally, tags, protein or oligonucleotides can be chemically conjugated to these peptides through ziyuBio. With many years of experience in providing modified peptide synthesis, we are your best choice for producing custom modified peptides on time and on budget. Our peptide modification services include but are not limited to the following:. ■ Fluorophores and Quenchers: Emitting and Quenching dyes for FRET and other dyes. ■ Attachment Chemistry: Linkers, Spacers and Polyethylene Glycol (PEG) modifications. ■ Unnatural Amino Acids: D-stereoisomers, Unnatural and special ...
To characterize the properties of the NvHsTRPA channel, we expressed it in HEK293 cells. Localization of the V5 and His epitope-tagged NvHsTRPA protein showed a staining pattern at the plasma membrane, demonstrating that it reaches to the cell surface (Fig. 5A). Nevertheless, some proteins are present in the cytoplasm as well. The molecular weight of the tagged protein was approximately 112 kDa, which is slightly larger than expected (105 kDa) from the amino acid sequence. The protein size was constant in the presence of tunicamycin, an inhibitor of N-glycosylation (Fig. 5B), indicating that NvHsTRPA does not contain N-glycans unlike the vertebrate TRPV1, 4, 5, and TRPC3 and 6 as reported [42]. The partial denature of NvHsTRPA (60°C for 5 min) may result in the aberrant migration through 6% SDS-PAGE gel which makes the correct estimation of protein size difficult. Nevertheless, the possibility that NvHsTRPA undergoes the other post-translational modifications can not be ruled out. Patch-clamp ...
Phosphorylation is one of the most important post-translational modification. Phosphorylation is involved in multiple biological process such as DNA damage and repair, transcrip- tion regulation, and metabolism. In human proteome, three types of major residue in the substrate; serine(S), threonine(T), and tyrosine (Y) is phosphorylated by their responsible kinase. Streaming motion of phosphorylation constructs an signal transaction network of the living cell.. Resent achievements in the area of mass spectrometry based proteomics accomplished in determining the phosphorylated residue of the protein in a massive scale. On the other hand, the global picture of signal network driven by phosphorylation still remains unclear, due to the lack of information of kinase-substrate pairs. To work out with this problem, computational approaches has been applied. While various numbers of computational predictors such as Scansite, NetworKIN, PPRED are available, there still remains a interruption in ...
We produce custom antibodies against PTMs such as phosphorylation, acetylation, and methylation as well as Ubiquitinylation or SUMOylation.
Forkhead box O3, also known as FOXO3 or FOXO3a, is a human protein encoded by the FOXO3 gene. FOXO3 belongs to the O subclass of the forkhead family of transcription factors which are characterized by a distinct fork head DNA-binding domain. There are three other FoxO family members in humans, FOXO1, FOXO4 and FOXO6. These transcription factors share the ability to be inhibited and translocated out of the nucleus on phosphorylation by proteins such as Akt/PKB in the PI3K signaling pathway (aside from FOXO6, which may be constitutively nuclear). Other post-translational modifications including acetylation and methylation are seen and can result in increased or altered FOXO3a activity. This protein likely functions as a trigger for apoptosis through upregulation of genes necessary for cell death, such as Bim and PUMA, or downregulation of anti-apoptotic proteins such as FLIP. Gopinath et al.(2014) demonstrate a functional requirement for FOXO3 as a regulator of Notch signaling pathway (an ...
Fibroblast Activation Protein (FAP) is a cellXsurface anchored dimeric protease, closely related to Dipeptidyl Peptidase (DPP) 4. This atypical serine protease has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a postXproline bond. FAP expression is difficult to detect in nonXdiseased adult organs, but is greatly up regulated in sites of tissue remodelling, which includes liver fibrosis, lung fibrosis, atherosclerosis, arthritis, tumours and embryonic tissues. Due to its restricted expression pattern and dual enzymatic activities, FAP is emerging as a unique therapeutic target. However, methods to exploit and target this protease are advancing more rapidly than knowledge of the fundamental biology of FAP. This thesis aims to rectify this imbalance, emphasising the need to better define the substrate repertoire and downstream effects of FAP enzyme activity to elucidate the role of this protease in biological and pathological processes. In this study, primary mouse ...
STATIN INHIBITION OF MACROPHAGE INTEGRIN-INDUCED RAC2-MYOSIN IIA INTERACTION: AN ANTI-INFLAMMATORY EFFECT. Kenneth E. Ike, Alan Morrison, and Jeffrey R. Bender. Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University, School of Medicine, New Haven, CT. HMG-CoA reductase inhibitors (statins) are pharmaceuticals that are utilized for the treatment of lipid disorders along with the primary and secondary prevention of coronary heart disease. HMG-CoA reductase is the rate-limiting enzyme in cholesterol synthesis, converting HMG-CoA to mevalonate. The isoprenoid products, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), are derived from the mevalonate pathway and serve as substrates in the prenylation of 2% of cellular proteins including the Rho family of low molecular weight G-proteins which mediate multiple cellular signals. Prenylation is an important post-translational modification of proteins that plays a role in the subcellular localization of proteins
Perform Glycan and Glycopeptide Qualitative Analysis, Glycan Structure Prediction & Multi stage Mass Spectrometry (MSn) data analysis using accurate ranking mechanism.
Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently ... read more described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. In contrast to other formats such as StageTips or batch incubations using TiO2 or Ti-IMAC beads, Fe-IMAC HPLC columns do not suffer from issues regarding incomplete phosphopeptide binding or elution and enrichment efficiency scales linearly with the amount of starting material. Here, we provide a step-by-step protocol for the entire phosphopeptide enrichment procedure including sample preparation (lysis, digestion, desalting), ...
Is this a trick question? :) Sudheendra Rao N R ,sudhee26 from gmail.com, wrote in message news:mailman.491.1202159543.2451.methods from net.bio.net... , Hi, , can somebody tell me what changes would occur to the molecular weight of a , protein being run in a denaturing PAGE after it undergoes Post , translational , modification? , like sumoylation can increase mol weight by 11kD. , , Sudheendra. , , -- , Think before agree , Think before you nod , but STOP thinking , and You Are God ...
Cytokines are a group of proteins and polypeptides that organisms use as signaling molecules. Most cytokines are glycoproteins less than 30 kDa in size and bind to specific, high-affinity cell surface receptors. Due to their central role in the immune system, cytokines are involved in a variety of immunological, inflammatory and infectious diseases and widely used in research, diagnostics and therapeutics. Cytokines generally alter the gene expression pattern of the target cell which leads to changes in the rate of cell proliferation and/or in the state of cell differentiation. Currently, these proteins are predominantly produced in non-human cells (e.g. E. coli, SF9, CHO) and therefore lack authenticity due to the absence of physiologically relevant glycosylation. In addition, a number of important cytokines are not commercially available due to inadequate proteolytic processing, protein folding or other post-translational modifications that do not occur in the non-human cell expression ...
H1S186ph. H1.4 serine 186 (reported as H1S187) phosphorylation is preferentially associated with active rDNA promoters and enriched at hormone response element (PMID: 20439994). ...
Authors: Georges Khoury, Talia M Mota, Shuang Li, Carolin Tumpach, Michelle Y Lee, Jonathan Jacobson, Leigh Harty, Jenny L Anderson, Sharon R Lewin, Damian FJ Purcell
C.Post-Translational Modification Proteomics--phosphoproteome identification Protein post-translational modification (PTM) increases the functional diversity of the proteome either by the covalent addition of chemical moieties or functional groups, or by the proteolytic cleavage of regulatory subunits or the degradation of protein complexes. Many large-scale post-translational modification studies have recently been performed on various organisms, and phosphorylation, acetylation, methylation, and glycosylation are among the most intensively studied PTM proteomes. Protein phosphorylation is an ubiquitous post-translational modification, essential to many physiological and biological processes and cellular events. Phosphoproteomic analysis provides identification of phosphorylated proteins and peptides, providing key data for understanding their biological functions. This proteomic method has greatly enhanced our understanding of cellular phosphoproteins and their dynamic regulatory mechanisms in ...
Ngn3 is recognized as a regulator of pancreatic endocrine formation, and Notch signaling as an important negative regulator Ngn3 gene expression. By conditionally controlling expression of Ngn3 in the pancreas, we find that these two signaling components are dynamically linked. This connection invol …
Nature provides an abundant source of functional proteins for designing new systems. To date, chromatin proteins are an untapped resource. A class of chromatin proteins, known as effectors, have the remarkable ability to discriminate and bind to specific post-translational modifications of target proteins called histones. Can a synthetic protein device be engineered to read histone modifications? Can we use this type of device as a new tool to monitor changes in histone modifications in single living cells? Accomplishing these goals will allow scientists to probe histone modification at unprecedented resolution, thus furthering our understanding of the dynamics of histone modifications associated with cancer and normal cell development. ...
E3 protein ligase that mediates ufmylation, the covalent attachment of the ubiquitin-like modifier UFM1 to substrate proteins, a post-translational modification on lysine residues of proteins that may play a crucial role in a number of cellular processes. Mediates DDRGK1 ufmylation and may regulate the proteasomal degradation of DDRGK1 and CDK5RAP3 thereby modulating NF-kappa-B signaling (PubMed:20018847, PubMed:20164180, PubMed:20228063, PubMed:25219498). May also through TRIP4 ufmylation play a role in nuclear receptors-mediated transcription (PubMed:25219498). May play a role in the unfolded protein response, mediating the ufmylation of multiple proteins in response to endoplasmic reticulum stress (PubMed:23152784).
YM-216391, an autitumor natural product, represents a new class of cyclic peptides containing a polyoxazolethiazole moiety, Herein we describe its gene cluster encoding the biosynthetic paradigm featuring a ribosomally synthesizing procursor peptide followed by a series of novel posttranslational modifications which include (1) cleavage of both N-terminal leader peptide and C-terminal extension peptide and cyclization in a head-to-tail fashion, (ii) conversion of an L-Ile to D-allo-Ile, and (iii) beta-hydroxylation of Phe by a P450 monooxygenase followed by further heterocyclization and oxidation to form a phenyloxazole moiety. The cluster was heterologously expressed in Streptomyces lividans to bypass difficult genetic manipulation. Deletion of the ymR3 gene, encoding a putative transcriptional regulator, increased the YM-216391 yield about 20-fold higher than the original yields for the heterologous expression of wild-type cluster, which set the stage for further combinatorial biosynthesis ...
The NF-κB family of transcription factors play a central role in the inducible expression ofinflammatory genes during the immune response, and the proper regulation of these genes is acritical factor in the maintenance of immune homeostasis. The chromatin environment atstimulus-responsive NF-κB sites is a major determinant in transcription factor binding, anddynamic alteration of the chromatin state to facilitate transcription factor binding is a keyregulatory mechanism. NF-κB is in turn able to influence the chromatin state through a variety ofmechanisms, including the recruitment of chromatin modifying co-activator complexes such asp300, the competitive eviction of negative chromatin modifications, and the recruitment ofcomponents of the general transcriptional machinery. Frequently, the selective interaction withthese co-activators is dependent on specific post-translational modification of NF-κB subunits.Finally, the mechanisms of inducible NF-κB activity in different immune cell types seem to
Nitro-fatty acids (NO(2)-FA) are electrophilic signaling mediators formed by reactions of nitric oxide and nitrite. NO(2)-FA exert anti-inflammatory signaling actions through post-translational protein modifications. We report that nitro-oleic acid (OA-NO(2)) stimulates proMMP-7 and proMMP-9 proteolytic activity via adduction of the conserved cysteine switch domain thiolate. Biotin-labeled OA-NO(2) showed this adduction occurs preferentially with latent forms of MMP, confirming a role for thiol alkylation by OA-NO(2) in MMP activation. In addition to regulating pro-MMP activation, MMP expression was modulated by OA-NO(2) via activation of peroxisome proliferator-activated receptor-γ. MMP-9 transcription was decreased in phorbol 12-myristate 13-acetate-stimulated THP-1 macrophages to an extent similar to that induced by the peroxisome proliferator-activated receptor-γ agonist Rosiglitazone. This was affirmed using a murine model of atherosclerosis, ApoE(-/-) mice, where in vivo OA-NO(2) administration
Lysine acetylation is a conserved, reversible, post-translational protein modification regulated by lysine acetyltransferases (KATs) and lysine deacetylases (KDACs; also known as histone deacetylases (HDACs)) that is involved in many cellular signalling pathways and diseases. Studies in animal models have revealed a regulatory role of reversible lysine acetylation in hypertension, vascular diseases, arrhythmia, heart failure and angiogenesis. Evidence from these studies indicates a therapeutic role of KDAC inhibitors (also known as HDAC inhibitors) in cardiovascular diseases. In this Review, we describe the diverse roles of KATs and KDACs in both the normal and the diseased heart. Among KDACs, class II and class III HDACs seem to have a protective role against both cardiac damage and vessel injury, whereas class I HDACs protect against vessel injury but have deleterious effects on the heart. These observations have important implications for the clinical utility of HDAC inhibitors as therapeutic agents
membrane, dolichyl-diphosphooligosaccharide-protein glycotransferase activity, co-translational protein modification, glycoprotein catabolic process, post-translational protein modification, protein N-linked glycosylation via asparagine, response to unfolded protein, ubiquitin-dependent ERAD pathway
1. Acetylation Databases. (1) PhosphoSitePlus: (PSP) is a comprehensive, manually curated and interactive resource on post-translational modifications (PTM). PSP contains encompasses 130000 non-redundant modification sites, manily on phosphorylation, ubiquitinylation and acetylation (Hornbeck, et al., 2004). (2) g2pDB: A Database Mapping Protein Post-Translational Modifications to Genomic Coordinates. The original data comes mainly from published studies, many of which involve the investigation of post-translational modification acceptor site assignments, e.g., phosphorylation, ubiquitination, SUMOylation, acetylation, and N-linked glycosylation sites. (Keegan S, et al., 2016). (3) dbPTM 2.0: integrates experimentally verified PTMs from several databases, and to annotate the predicted PTMs on Swiss-Prot proteins , 2,071 acetylation sites were included while most of which were N-alpha-terminal ones (Lee TY, et al., 2006) . (4) HPRD release 9: HPRD currently contains information for 16,972 PTMs ...
Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be ...
Protein ubiquitination is an important post-translational modification involved in several essential signalling pathways. It has different effects on the target protein substrate, i.e., it can trigger the degradation of the protein in the proteasome, change the interactions of the modified protein with its partners, or affect its localization and activity. In order to understand the molecular mechanisms underlying the consequences of protein ubiquitination, scientists have to face the challenging task of producing ubiquitinated proteins for structural characterization with X-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy. These techniques require milligrams of homogeneous samples of high purity. The strategies proposed so far for the production of ubiquitinated proteins can be divided into two groups, i.e., chemical (or non-enzymatic) and enzymatic methodologies. In this review, we summarize the still very sparse examples available in the literature that describe successful
The conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates is an important post-translational modification that has ramifications for cancer and other diseases. As the sole E2 enzyme in the tightly regulated E1/E2/E3 SUMOylation enzymatic cascade, Ubc9 plays a central role in the conjugation of all three SUMO isoforms to a variety of protein targets. Although Ubc9 is viewed as a promising anti-cancer drug target, the development of small-molecule Ubc9 inhibitors has proven to be very difficult. In the past decade, fragment-based drug design has emerged as a powerful approach to identify ligands for challenging protein targets that can provide excellent starting points for the development of potent inhibitors. By X-ray crystallographic fragment screening, we have identified two small-molecule fragments that bind to Ubc9 at a location that is distal from its active site. Although these fragments have weak affinity for Ubc9, biochemical assays have confirmed that they inhibit ...
In eukaryotes, the conjugation of the ubiquitinlikeprotein NEDD8 onto protein targets is an important post-translational modification. The best understood neddylation targets are the cullins, scaffold subunits of E3 ubiquitin ligases, where neddylation as well as deneddylation, facilitated by the protease activity of the CSN (COP9 signalosome), are required to control ubiquitin ligase assembly, function and ultimately substrate degradation. Little is known about the role of other deneddylating enzymes besides CSN and the role of neddylation and deneddylation of their substrates. We previously characterized Arabidopsis thaliana mutants with defects in the conserved NEDD8-specific protease DEN1 (DENEDDYLASE1). These mutants display only subtle growth phenotypes despite the strong accumulation of a broad range of neddylated proteins. Specifically, we identified AXR1 (AUXIN RESISTANT1), a subunit of the heterodimeric NAE (E1 NEDD8 ACTIVATING ENZYME), as highly neddylated in den1 mutants. Here, we ...
Chumpen Ramirez S et at, Biochem J., 2017. Ganglioside glycosyltransferases (GGTs) are type-II membrane proteins bearing a short N-terminal cytoplasmic tail, a transmembrane domain (TMD), and a lumenal catalytic domain. The expression and activity of these enzymes largely determine the quality of the glycolipids that decorate mammalian cells membranes. Many glycosyltransferases are themselves glycosylated and this is important for their proper localisation, but few if any other post-translational modifications of these proteins have been reported. Here we show that the GGTs, ST3Gal-V, ST8Sia-I, and β4GalNAcT-I are S-acylated at conserved cysteine residues located close the cytoplasmic border of their TMDs. ST3Gal-II, a GT that sialylates glycolipids and glycoproteins, is also S-acylated at a conserved cysteine located in the N-terminal cytoplasmic tail. Many others GTs also possess cysteine residues in their cytoplasmic regions suggesting that this modification occurs on these GTs as well. ...
TY - JOUR. T1 - Protein phosphorylation detection using dual-mode field-effect devices and nanoplasmonic sensors. AU - Bhalla, Nikhil. AU - Di Lorenzo, Mirella. AU - Pula, Giordano. AU - Estrela, Pedro. PY - 2015/3/3. Y1 - 2015/3/3. N2 - Phosphorylation by kinases is an important post-translational modification of proteins. It is a critical control for the regulation of vital cellular activities, and its dysregulation is implicated in several diseases. A common drug discovery approach involves, therefore, time-consuming screenings of large libraries of candidate compounds to identify novel inhibitors of protein kinases. In this work, we propose a novel method that combines localized surface plasmon resonance (LSPR) and electrolyte insulator semiconductor (EIS)-based proton detection for the rapid identification of novel protein kinase inhibitors. In particular, the selective detection of thiophosphorylated proteins by LSPR is achieved by changing their resonance properties via a pre-binding with ...
This thesis deals with an important post-translational modification, ubiquitination. An important part of ubiquitin, regulatory protein, is the N-terminus and seven lysine residues, through which originates mutual binding of ubiquitin molecules in the polyubiquitin chain. The various possibilities of binding provide diversity in the creation of polyubiquitin chains, and thus their significance. Since ubiquitinated proteins in the analyzed protein mixtures exist mostly as a minor component, the first important and difficult step is their correct separation and identification. Experimental part of this work deals with precisely this part of the research and thus contains optimization methods for isolating and identifying of ubiquitin proteins by antibodies on the peptide and protein level. It has been shown that the optimization takes more time and work, thus future progressin this area is expected. …víceméně ...
Abnormal levels of cross-linking in fibrillar collagen strands have been shown to cause a number of human and animal diseases. Cross-linking is a vital step in fibrillogenesis and contributes greatly to the structural integrity of collagenous tissues. Conversely, defects in cross-link formation can significantly alter fibrillar organisation and lead to pathogenesis. Because collagen cross-links form on collagen-specific hydroxylated lysine residues, an understanding of the link between hydroxylysine and cross-link concentrations is needed to determine whether the level of hydroxylysine, the stereochemistry of these hydroxylysine residues, or other post-translational modifications such as glycosylation affect the level of cross-linking in tissue. While some research has been done to elucidate the connection between the two in different tissue types from the same animal, little has been undertaken to relate hydroxylation and glycosylation of lysine and hydroxylysine to the concentration and types ...
Multiple biotic and abiotic environmental factors may constitute stresses that affect plant growth and yield in crop species. Advances in plant physiology, genetics, and molecular biology have greatly improved our understanding of plant responses to stresses. This book details on technologies that have emerged during the past decade and have been useful in studying the multigenicity of the plant abiotic stress response. Upstream molecular mechanisms are involved in the plant response to abiotic stress, above all in the regulation of timings and amount of specific stress responses. Post-transcriptional mechanisms based on alternative splicing and RNA processing, as well as RNA silencing define the actual transcriptome supporting the stress response. Beyond protein phosphorylation, other post-translational modifications like ubiquitination and sumoylation regulate the activation of pre-existing molecules to ensure a prompt response to stress factors. The text in this book deals with the importance ...
This service allows the production of antibodies that will specifically recognize the phosphorylated state of your target protein. The approach includes the synthesis and purification of phosphorylated and non-phosphorylated peptides, immunisation, fabrication of two affinity columns for successive purification. Extensive ELISA on phospho-specific antibodies as well as on the fraction specific to the non-phosphorylated state is performed. We have successfully utilized this method with other post-translational modifications including acetylation and methylation. ...
Pharmacia Corporation, Milan, Italy is seeking talented people to join our research groups in a multidisciplinary and highly interactive environment. Pharmacia Corporation is a global company with global ambitions. We offer competitive salaries, benefits and global career opportunities limited only by your skill, creativity and performance. If you are interested in any of the positions described, please send a CV to Dr. Antonella Isacchi Phone 0248383791 Email Antonella.Isacchi at eu.pnu.com or Dr Simon Plyte simon.plyte at eu.pnu.com Open positions list - Biology Department Protein Chemist/Mass Spectrometrist The candidate will be responsible for a Protein Chemistry Unit of 4-5 scientists focused on the analytical characterization of recombinant proteins to be used for x-ray crystallographic and NMR studies. These efforts involve domain mapping by limited proteolysis, N- and C- terminal sequence analysis, and the mapping of phosphorylation and other post-translational modifications by LCMS and ...
FIGURE 3. The reduced form of glutathione (GSH) acting as a double-edged sword. As illustrated, GSH is a major cellular defense against oxidative and electrophilic stress. It also plays an important role in redox signaling. On contrary, this tri-peptide, under certain conditions, make the host cell or organism more susceptible to such conditions as anxiety, pathogen infections, and tumorigenesis.. 3.1.4. Protein Deglutathionylation Reversible protein S-glutathionylation (protein-SSG) is an important post-translational modification involved in redox signaling. Analogous to protein dephosphorylation catalyzed by phosphatases, glutaredoxin using GSH as a cofactor/electron donor catalyzes deglutathionylation of proteins. This participates in regulating diverse intracellular signaling pathways [6, 7].. 3.2. Atypical Biological Activities. 3.2.1. Anxiety. In addition to the above well-established functions, GSH also possesses atypical biological activities, including both beneficial and detrimental ...
back to overview Galactose is a monosaccharide (sugar). Galactosylation (especially O-galactosylation at Ser and Thr) is an important post-translational modification of proteins ...
Linear ubiquitination is definitely a important posttranslational adjustment that regulates immune system signaling and cell death pathways, notably tumor necrosis element receptor 1 (TNFR1) signaling. caspase\8 or epidermal mutilation of FADD.21, 23 These studies collectively corroborate a central part of LUBAC in restraining aberrant service of TNFR1\induced cell death machineries in order to maintain cells homeostasis. Although mice show liver swelling, it remains unfamiliar which cells and cell types contribute to hepatitis. In addition, the physiological part of LUBAC in LPCs remains unfamiliar. Here, we looked into the part of linear ubiquitination and Rabbit Polyclonal to MMP-19 LUBAC in liver swelling and carcinogenesis by studying mice that lack HOIP, the central and catalytically active component of LUBAC, specifically in LPCs. Materials and Methods ANIMALS All animal studies were carried out relating to an appropriate license under the Animals (Scientific TSA Methods) Take action of ...
Les histones sont des protéines nucléaires hautement conservées chez les cellules des eucaryotes. Elles permettent dorganiser et de compacter lADN sous la forme de nucléosomes, ceux-ci representant les sous unités de base de la chromatine. Les histones peuvent être modifiées par de nombreuses modifications post-traductionnelles (PTMs) telles que lacétylation, la méthylation et la phosphorylation. Ces modifications jouent un rôle essentiel dans la réplication de lADN, la transcription et lassemblage de la chromatine. Labondance de ces modifications peut varier de facon significative lors du developpement des maladies incluant plusieurs types de cancer. Par exemple, la perte totale de la triméthylation sur H4K20 ainsi que lacétylation sur H4K16 sont des marqueurs tumoraux spécifiques a certains types de cancer chez lhumain. Par conséquent, létude de ces modifications et des événements determinant la dynamique des leurs changements dabondance sont des atouts importants ...
For this analysis they are primarily using an LTQ-Orbitrap XL with ETD and employing both CID and ETD fractionation. Surprisingly, the majority of the information being obtained for PTM matches is not coming from the ETD. This is likely due to the lower speed/efficiency of the earliest ETD system compared to the ones I normally get to mess around with. It does, however, contribute meaningfully to the study. This is a nice clear study but I mostly highlight it here because Im very interested in what they are going to do next AND how this data is going to line up with other well established histone PTM datasets we have from other models. So...this post is kind of to remind myself to check back on these guys later...sorry ...
Small ubiquitin-related modifier 2; Ubiquitin-like protein that can be covalently attached to proteins as a monomer or as a lysine-linked polymer. Covalent attachment via an isopeptide bond to its substrates requires prior activation by the E1 complex SAE1-SAE2 and linkage to the E2 enzyme UBE2I, and can be promoted by an E3 ligase such as PIAS1-4, RANBP2, CBX4 or ZNF451. This post-translational modification on lysine residues of proteins plays a crucial role in a number of cellular processes such as nuclear transport, DNA replication and repair, mitosis and signal transduction. Polyme [...] (95 aa ...
Protein phosphorylation is arguably the most important reversible post-translational modification that occurs in cells. Protein kinases and phosphatases add or remove, respectively, phosphate groups that regulate enzymatic activity, protein-protein interactions, and protein tertiary structures. Knowledge of the sites of phosphorylation on a particular protein can aid in understanding the mechanism by which that protein affects a biological pathway.. A variety of techniques are available for detecting phosphorylated peptides. Traditional methods such as TLC and Edman sequencing have the disadvantages that they typically require the use of radioactively labeled sample and either prior knowledge of the protein sequence or the ability to make some assumptions about the peptide sequence. Proteins can be labeled in vivo using [32P]orthophosphate or in vitro using purified kinase and [γ-32P]ATP. The phosphorylated proteins are enzymatically digested, and the phosphopeptides are isolated by high ...
Antibody-based Therapeutics N-linked glycosylation is a common post-translational modification on many antibody-based therapeutics, and has been linked to safety, stability and activity. Modification should therefore be monitored and controlled.During this webcast, our expert Michael Walker discusses peptide mapping and middle-up mass spectrometry as characterization techniques that give relative levels of different glycoforms at specific sites, and compares to more common methodologies of enzymatic stripping and fluorescent labelling.
ADP-ribosylation is the addition of one or more ADP-ribose moieties to a protein. It is a reversible post-translational modification that is involved in many cellular processes, including cell signaling, DNA repair, gene regulation and apoptosis. Improper ADP-ribosylation has been implicated in some forms of cancer. It is also the basis for the toxicity of bacterial compounds such as cholera toxin, diphtheria toxin, and others. The first suggestion of ADP-ribosylation surfaced during the early 1960s. At this time, Pierre Chambon and coworkers observed the incorporation of ATP into hen liver nuclei extract. After extensive studies on the acid insoluble fraction, several different research laboratories were able to identify ADP-ribose, derived from NAD+, as the incorporated group. Several years later, the enzymes responsible for this incorporation were identified and given the name poly (ADP-ribose) polymerase. Originally, this group was thought to be a linear sequence of ADP-ribose units ...
The small ubiquitin related modifier (SUMO)-mediated posttranslational protein modification is widely conserved among eukaryotes. Similar to ubiquitination, SUMO modifications are attached to the substrate protein through three reaction steps by the E1, E2 and E3 enzymes. To date, multiple families of SUMO E3 ligases have been reported in yeast and animals, but only two types of E3 ligases have been identified in Arabidopsis: SAP and Miz 1 (SIZ1) and Methyl Methanesulfonate-Sensitivity protein 21 (MMS21)/HIGH PLOIDY 2 (HPY2), hereafter referred to as HPY2. Both proteins possess characteristic motifs termed Siz/PIAS RING (SP-RING) domains, and these motifs are conserved throughout the plant kingdom. Previous studies have shown that loss-of-function mutations in HPY2 or SIZ1 cause dwarf phenotypes and that the phenotype of siz1-2 is caused by the accumulation of salicylic acid (SA). However, we demonstrate here that the phenotype of hpy2-1 does not depend on SA accumulation. Consistently, the expression
SUMO1 is a member of a ubiquitin-like group of enzymes responsible for SUMOylation, a type of reversible posttranslational protein modification (82, 100). SUMOylation facilitates a wide array of cellular homeostasis-related functions, such as nuclear translocation, cell signaling, and protein allocation (28). Furthermore, SUMOylation regulates tumor suppressors such as PTEN (6), p53 (99), and VHL (18) and promotes cell survival, via the aforementioned HDAC2-dependent p53 acetylation (5) and its partaking in the DDR (81). PARK7 is both a target for SUMOylation (109) and a regulator of the SUMO-1 pathway, inhibiting SUMOylation unless functionally impaired by oxidation (56). A PARK7-induced dysregulation of SUMOylation could lead to increased survival of neoplastic cells through either the SUMO1 or PARK7 pathway, given that they appear to be intertwined. This is a speculation, however, and more specific studies should address it. Still, no published reports on the potential role of SUMO1 exist on ...
TY - JOUR. T1 - Post-translational modifications as key regulators of apicomplexan biology. T2 - insights from proteome-wide studies. AU - Yakubu, Rama R.. AU - Weiss, Louis M.. AU - Silmon de Monerri, Natalie C.. PY - 2018/1. Y1 - 2018/1. N2 - Parasites of the Apicomplexa phylum, such as Plasmodium spp. and Toxoplasma gondii, undergo complex life cycles involving multiple stages with distinct biology and morphologies. Post-translational modifications (PTMs), such as phosphorylation, acetylation and glycosylation, regulate numerous cellular processes, playing a role in every aspect of cell biology. PTMs can occur on proteins at any time in their lifespan and through alterations of target protein activity, localization, protein-protein interactions, among other functions, dramatically increase proteome diversity and complexity. In addition, PTMs can be induced or removed on changes in cellular environment and state. Thus, PTMs are likely to be key regulators of developmental transitions, biology ...
The folding, quality control, and secretion of collagen presents a significant challenge to collagen-producing cells. Each monomeric polypeptide must undergo extensive post-translational modifications, folding and assembly of the C-terminal propeptide globular domain, and isomerization of hundreds of prolyl bonds into the trans conformation before the mature triple helix can form. The triple-helical domain lacks a traditional hydrophobic core that often drives the assembly and folding of globular proteins. Even once folded, the triple helix is at best marginally stable at body temperature and is prone to local regions of unwinding. The process must be highly orchestrated by the endoplasmic reticulums chaperone network, including maintaining newly synthesized collagen polypeptides in an unfolded, non-aggregated, and unassembled form until after the extreme C-terminus adopts its folded structure. Despite decades of work, however, the mechanisms of collagen folding remain poorly understood, and ...
Background: Ubiquitination is a vital posttranslational protein modification involved in the regulation of many eukaryotic signalling pathways. Aberrant ubiquitin signalling is known to be a molecular causality of certain cancer, neurodegenerative, immune system or cardiovascular diseases. The recent development of mass spectrometry methods enables qualitative and quantitative ubiquitination...
Human bone sialoprotein (BSP) is an essential component of the extracellular matrix of bone. It is thought to be the primary nucleator of hydroxyapatite crystallization, and is known to bind to hydroxyapatite, collagen, and cells. Mature BSP shows extensive post-translational modifications, including attachment of glycans, sulfation, and phosphorylation, and is highly flexible with no specific 2D or 3D structure in solution or the solid state. These features have severely limited the experimental characterization of the structure of this protein. We have therefore developed a 3D structural model for BSP, based on the available literature data, using molecular modelling techniques. The complete model consists of 301 amino acids, including six phosphorylated serines and two sulfated tyrosines, plus 92 N- and O-linked glycan residues. A notable feature of the model is a large acidic patch that provides a surface for binding Ca2+ions. Density functional theory quantum calculations with an implicit ...
Soil-dwelling Streptomyces bacteria such as S.coelicolor have to constantly adapt to the nitrogen (N) availability in their habitat. Thus, strict transcriptional and post-translational control of the N-assimilation is fundamental for survival of this species. GlnR is a global response regulator that controls transcription of the genes related to the N-assimilation in S. coelicolor and other members of the Actinomycetales. GlnR represents an atypical orphan response regulator that is not activated by the phosphorylation of the conserved aspartate residue (Asp 50). We have applied transcriptional analysis, LC-MS/MS analysis and electrophoretic mobility shift assays (EMSAs) to understand the regulation of GlnR in S. coelicolor M145. The expression of glnR and GlnR-target genes was revisited under four different N-defined conditions and a complex N-rich condition. Although, the expression of selected GlnR-target genes was strongly responsive to changing N-concentrations, the glnR expression itself ...
Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B and U1-C and that their
Dr. Mamulas received degrees from UCLA, the University of Notre Dame and the University of Oklahoma. Dr. Mamulas central research interests are in investigating the early events involved with breaking immune tolerance to self proteins, both in autoimmune disease and in tumor biology. Overall, it is the goal of Dr. Mamulas laboratory to understand the mechanisms that may shift this balance toward the initiation of anti-self immune responses. Seminal work from the Mamula lab elucidated the biochemical forms of autoantigens capable of breaking immunologic tolerance to intracellular autoantigens in systemic lupus erythematosus (SLE), and type 1 diabetes (T1D). Simply put, Dr. Mamula examines posttranslational protein modifications that alter cellular biology and immunity. These studies have now been applied to the development of novel therapeutic approaches in developing anti-tumor vaccines in breast cancer and colon cancer. In addition, studies from the Mamula laboratory first demonstrated the ...
Dr. Mamulas received degrees from UCLA, the University of Notre Dame and the University of Oklahoma. Dr. Mamulas central research interests are in investigating the early events involved with breaking immune tolerance to self proteins, both in autoimmune disease and in tumor biology. Overall, it is the goal of Dr. Mamulas laboratory to understand the mechanisms that may shift this balance toward the initiation of anti-self immune responses. Seminal work from the Mamula lab elucidated the biochemical forms of autoantigens capable of breaking immunologic tolerance to intracellular autoantigens in systemic lupus erythematosus (SLE), and type 1 diabetes (T1D). Simply put, Dr. Mamula examines posttranslational protein modifications that alter cellular biology and immunity. These studies have now been applied to the development of novel therapeutic approaches in developing anti-tumor vaccines in breast cancer and colon cancer. In addition, studies from the Mamula laboratory first demonstrated the ...
The members of the recently established DFG Research Training Group 2155 (ProMoAge) gathered for their first meeting in the historical city of Wittenberg during the weekend from 5th to 7th November 2016. What better location for this kick-off-meeting could be chosen than the „Leucorea, which was founded over 500 years ago and has later merged with the Friedrichs University of Halle to form the Martin Luther University Halle-Wittenberg. The aim of the meeting was to introduce each specific subproject of ProMoAge and the working group behind it. 14 innovative projects dealing with posttranslational protein modifications and their role in ageing were shortly presented and discussed with enthusiasm by PhD and MD students, postdocs and group leaders. Guest lectures about good scientific practice, ProMoAge-related epidemiological cohort studies and newest insights in proteasome modulation during ageing complemented the diverse programme. One should not stay in Wittenberg without visiting the ...
Author summary Ubiquitin and ubiquitin-like post-translational modifications are evolutionarily conserved and involved in fundamental cellular processes essential to all eukaryotes. As such, enzymatic components of these pathways present attractive targets for therapeutic intervention for both chronic and communicable diseases. Nedd8 modification of cullin ubiquitin E3 ligases is critical to the viability of eukaryotic organisms and mediates cell cycle progression and DNA damage repair. Given the complex lifecycle and unusual replication mechanisms of the malaria parasite, one would expect neddylation to be of central importance to its survival, yet little is known about this pathway in Plasmodium. Here we present our findings on how Nedd8 removal is controlled in Plasmodium falciparum and how this pathway differs to that of its human host.
Our laboratory located at Cystic Fibrosis and Pulmonary Research Center. One of the main focuses of our work is the characterization of the large mucin gene products (Mr 2-3 million) and the complexes they make (Mr 10-100 million) essential for the formation of the mucus gels vital for epithelial protection and function. our current work is focused around the human lung where there are many hypersecretory human diseases including asthma, cystic fibrosis, and chronic bronchitis in which these glycoconjugates are centrally implicated. My proteomic interest includes designing and implementing novel mass spectral methods for identifying proteins and glycoproteins in mucus gels where conventional proteomic methods do not work. Many of the molecules are heavily modified and thus MS methods for characterizing post-translational modifications are vital in this work. ...
An elevated level of LDL cholesterol is associated with the development of atherosclerosis and is also associated with aging. Modification of LDL particle is one of the main contributors to the development of atherosclerosis and is elevated with increasing plasma LDL concentrations. Modified LDL is usually composed of LOOH/aldehydes, unfolded protein and some protein post-translational modifications. It has been debated whether the lipid peroxides or unfolded apoB-100 protein is important. An important pathway in atherosclerosis may be the phosphorylation of JNK-2 in ECs. OxLDL-R CD-36 knockout macrophages which have decreased foam cell formation and decreased JNK-2 phosphorylation as well as an ApoE and JNK-2 double knockout mouse has decreased lesion size and MFC formation. Foam cells have increased ROS production and mitochondria are the major source of ROS and this evidence may suggest that p-JNK-2 is involved in regulating mitochondrial function.; We hypothesize that ONOO- induced nitration ...
The Discovery Award in Proteomic Sciences has been awarded by the Human Proteome Organization (HUPO) to Albert J.R. Heck, Professor at the Science Faculty of Utrecht University. Heck is also scientific director of the Netherlands Proteomics Centre and coordinator of the European proteomics infrastructure PRIME-XS and the NWO roadmap funded Large-Scale Proteomics Research Facility Proteins At Work.. The Discovery in Proteomic Sciences award recognizes a scientist for an eminent single discovery in the field of proteomics. Heck receives the award because he has implemented innovative mass spectrometric methods with a unique emphasis on protein post-translational modifications and interactions.. Heck will receive the award during a ceremony at the HUPO World Congress in Yokohama, Japan, September 18, 2013.. Past award recipients are amongst others: Carol Robinson, Steve Carr, Matthias Mann, Catherine Costello, John Yates, Ruedi Aebersold and Brian Chait.. ...
Glycoscience is gaining recognition as an important component of life science, as emphasized in two recently published roadmaps issued by the American National Research Council in 2012 (1) and by the European Science Foundation (ESF) GlycoForum (http://ibcarb.com/wp-content/uploads/A-roadmap-for-Glycoscience-in-Europe.pdf) in 2015. Both references point to the same need for organizing access to glycan-related data that is absent in current bioinformatics resources.. Glycosylation is the most common protein post-translational modification yet its role is far from being understood. Glycans, proteins to which they are attached (glycoproteins) and proteins to which they bind (lectins or carbohydrate-binding proteins) are the main molecular actors in this overall cell surface picture as well as the enzymes that are needed to synthesize or trim the attached glycans. The first challenge in collecting this data is the wide range of experimental techniques used to analyze glycans and to elucidate their ...
Photosynthetic electron transport and biological energy transduction are studied by electron spin resonance and time-resolved optical and electroabsorption spectroscopies. Biochemical and biophysical research also focuses on the mechanisms of protein folding and aggregation, protein folding defects related to human diseases, and the molecular structures of proteins, including amino acid sequence determination and identification of protein post-translational modifications. Carbohydrate biochemistry and glycobiology are used to understand disease processes and to develop new therapeutic agents. The biochemical aspects of biotechnology including chemoenzymatic synthesis, biocatalysis, and metabolic engineering are being explored. The methodologies used include kinetic and spectroscopic analysis (NMR, fluorescence, circular dichroism, surface plasmon resonance (SPR) and FTIR of protein conformational changes), molecular modeling, computational graphics, and molecular mechanics calculations on ...
About the project There are hundreds of types of protein post-translational modification (PTM), which alter protein structure, function, stability, etc.
HIstome is a freely available, specialist, electronic database dedicated to display infomation about human histone variants, sites of their post-translational modifications and about various histone modifying enzymes. HIstome data is available by browsing the contents. The database covers 5 types of histones, 8 types of their post-translational modifications and 13 classes of modifying enzymes. Current version contains information for about ~50 histone proteins and ~150 histone modifying enzymes. Many data fields are hyperlinked to other databases (e.g. UnprotKB/Swiss-Prot, HGNC, OMIM, Unigene etc.). Additionally, this database also provides sequences of promoter regions (-700 TSS +300) for all gene entries. These sequences were extracted from the UCSC genome browser. Sites of post-translational modifications of histones were manually searched from PubMed listed literature. This infobase would be of use to biology researchers, especially those interested in epigenetic regulation. We welcome ...
A system-wide analysis of cell signaling requires detecting and quantifying many different proteins and their posttranslational modification states in the same cellular sample. Here, we present Protocols for two miniaturized, array-based methods, one of which provides detailed information on a central signaling protein and the other of which provides a broad characterization of the surrounding signaling network. We describe a bead-based array and its use in characterizing the different forms and functions of β-catenin, as well as lysate microarrays (reverse-phase protein arrays) and their use in detecting and quantifying proteins involved in the canonical and noncanonical Wnt signaling pathways. As an application of this dual approach, we characterized the state of β-catenin signaling in cell lysates and linked these molecule-specific data with pathway-wide changes in signaling. The Protocols described here provide detailed instructions for cell culture methods, bead arrays, and lysate ...
Monoclonal antibody and Fc fusion protein drugs are complex heterogeneous mixtures of numerous different protein variants and modifications. N-glycosylation as one of the most complex post translational modification influences the structural characteristics of the antibodies Fc part thereby potentially modulating effector function and pharmacokinetics. Several investigations on the relationship between N-glycosylation and pharmacokinetics have been published. However, this structure-function relationship is not fully understood. In this review potential alterations with focus on N-glycosylation of mAbs and Fc fusion proteins and the possible effects on the pharmacokinetics are reviewed and the current understandings of the underlying mechanisms are described. (C) 2016 The Authors. Published by Elsevier B.V. ...