Modification of calmodulin on Lys-75 by carbamoylating nitrosoureas

This paper describes characterization of the reaction of calmodulin with a series of nitrosoureas which are capable of releasing amine-reactive isocyanates of varying hydrophobic character. The site of calcium-dependent carbamoylation on calmodulin by the antineoplastic agent 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (methyl CCNU) was determined to be Lys-75 as demonstrated using [ring-14C]methyl CCNU and sequence analysis of the sole labeled peptide obtained from tryptic digestion of reversed-phase high pressure liquid chromatography (HPLC)-purified radiolabeled calmodulin. CCNU, the 4-desmethylcyclohexyl derivative of methyl CCNU, and its reactive hydrolysis product, cyclohexyl isocyanate, were also determined to modify calmodulin in a similar manner and at the same site, as demonstrated by specific blockade of modification by the calmodulin antagonist calmidazolium. Nitrosoureas which release the less hydrophobic 4-hydroxy- and 4-carboxycyclohexyl isocyanates are unable to modify calmodulin at 25-fold higher concentrations than those required for modification with methyl CCNU, CCNU, or cyclohexyl isocyanate. With this monomodified Lys-75 derivative, purified to homogeneity by HPLC, differential effects of modification on the activation of bovine brain 3',5'-cyclic nucleotide phosphodiesterase (phosphodiesterase) and human erythrocyte Ca2+,Mg2+-ATPase were observed. Compared to the amounts of native calmodulin needed, phosphodiesterase required 7-fold higher amounts of this derivative to reach maximal activation, whereas the activation of the ATPase was unaffected. Clearly, different regions of calmodulin are responsible for the activation of phosphodiesterase and the ATPase. We conclude that Lys-75 is not essential for the function of calmodulin but is in a region of the molecule involved in interaction with phosphodiesterase as well as the binding of certain hydrophobic calmodulin antagonists.     

Inhibition by active site directed covalent modification of human glyoxalase I

The glyoxalase pathway is responsible for conversion of cytotoxic methylglyoxal (MG) to d-lactate. MG toxicity arises from its ability to form advanced glycation end products (AGEs) on proteins, lipids and DNA. Studies have shown that inhibitors of glyoxalase I (GLO1), the first enzyme of this pathway, have chemotherapeutic effects both in vitro and in vivo, presumably by increasing intracellular MG concentrations leading to apoptosis and cell death. Here, we present the first molecular inhibitor, 4-bromoacetoxy-1-(S-glutathionyl)-acetoxy butane (4BAB), able to covalently bind to the free sulfhydryl group of Cys60 in the hydrophobic binding pocket adjacent to the enzyme active site and partially inactivate the enzyme. Our data suggests that partial inactivation of homodimeric GLO1 is due to the modification at only one of the enzymatic active sites. Although this molecule may have limited use pharmacologically, it may serve as an important template for the development of new GLO1 inhibitors that may combine this strategy with ones already reported for high affinity GLO1 inhibitors, potentially improving potency and specificity.     

ROSETTALIGAND: protein-small molecule docking with full side-chain flexibility

Protein-small molecule docking algorithms provide a means to model the structure of protein-small molecule complexes in structural detail and play an important role in drug development. In recent years the necessity of simulating protein side-chain flexibility for an accurate prediction of the protein-small molecule interfaces has become apparent, and an increasing number of docking algorithms probe different approaches to include protein flexibility. Here we describe a new method for docking small molecules into protein binding sites employing a Monte Carlo minimization procedure in which the rigid body position and orientation of the small molecule and the protein side-chain conformations are optimized simultaneously. The energy function comprises van der Waals (VDW) interactions, an implicit solvation model, an explicit orientation hydrogen bonding potential, and an electrostatics model. In an evaluation of the scoring function the computed energy correlated with experimental small molecule binding energy with a correlation coefficient of 0.63 across a diverse set of 229 protein- small molecule complexes. The docking method produced lowest energy models with a root mean square deviation (RMSD) smaller than 2 A in 71 out of 100 protein-small molecule crystal structure complexes (self-docking). In cross-docking calculations in which both protein side-chain and small molecule internal degrees of freedom were varied the lowest energy predictions had RMSDs less than 2 A in 14 of 20 test cases.     

Myeloperoxidase-induced modification of HDL by isolevuglandins inhibits paraoxonase-1 activity

Reduced activity of paraoxonase 1 (PON1), a high-density lipoprotein (HDL)-associated enzyme, has been implicated in the development of atherosclerosis. Post-translational modifications of PON1 may represent important mechanisms leading to reduced PON1 activity. Under atherosclerotic conditions, myeloperoxidase (MPO) is known to associate with HDL. MPO generates the oxidants hypochlorous acid and nitrogen dioxide, which can lead to post-translational modification of PON1, including tyrosine modifications that inhibit PON1 activity. Nitrogen dioxide also drives lipid peroxidation, leading to the formation of reactive lipid dicarbonyls such as malondialdehyde and isolevuglandins, which modify HDL and could inhibit PON1 activity. Because isolevuglandins are more reactive than malondialdehyde, we used in vitro models containing HDL, PON1, and MPO to test the hypothesis that IsoLG formation by MPO and its subsequent modification of HDL contributes to MPO-mediated reductions in PON1 activity. Incubation of MPO with HDL led to modification of HDL proteins, including PON1, by IsoLG. Incubation of HDL with IsoLG reduced PON1 lactonase and antiperoxidation activities. IsoLG modification of recombinant PON1 markedly inhibited its activity, while irreversible IsoLG modification of HDL before adding recombinant PON1 only slightly inhibited the ability of HDL to enhance the catalytic activity of recombinant PON1. Together, these studies support the notion that association of MPO with HDL leads to lower PON1 activity in part via IsoLG-mediated modification of PON1, so that IsoLG modification of PON1 could contribute to increased risk for atherosclerosis, and blocking this modification might prove beneficial to reduce atherosclerosis.     

Phosphorylation of TET Proteins Is Regulated via O-GlcNAcylation by the O-Linked N-Acetylglucosamine Transferase (OGT)

TET proteins oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine and thus provide a possible means for active DNA demethylation in mammals. Although their catalytic mechanism is well characterized and the catalytic dioxygenase domain is highly conserved, the function of the regulatory regions (the N terminus and the low-complexity insert between the two parts of the dioxygenase domains) is only poorly understood. Here, we demonstrate that TET proteins are subject to a variety of post-translational modifications that mostly occur at these regulatory regions. We mapped TET modification sites at amino acid resolution and show for the first time that TET1, TET2, and TET3 are highly phosphorylated. The O-linked GlcNAc transferase, which we identified as a strong interactor with all three TET proteins, catalyzes the addition of a GlcNAc group to serine and threonine residues of TET proteins and thereby decreases both the number of phosphorylation sites and site occupancy. Interestingly, the different TET proteins display unique post-translational modification patterns, and some modifications occur in distinct combinations. In summary, our results provide a novel potential mechanism for TET protein regulation based on a dynamic interplay of phosphorylation and O-GlcNAcylation at the N terminus and the low-complexity insert region. Our data suggest strong cross-talk between the modification sites that could allow rapid adaption of TET protein localization, activity, or targeting due to changing environmental conditions as well as in response to external stimuli.     

Proteome-wide profiling of carbonylated proteins and carbonylation sites in HeLa cells under mild oxidative stress conditions

A number of oxidative protein modifications have been well characterized during the past decade. Presumably, reversible oxidative posttranslational modifications (PTMs) play a significant role in redox signaling pathways, whereas irreversible modifications including reactive protein carbonyl groups are harmful, as their levels are typically increased during aging and in certain diseases. Despite compelling evidence linking protein carbonylation to numerous disorders, the underlying molecular mechanisms at the proteome remain to be identified. Recent advancements in analysis of PTMs by mass spectrometry provided new insights into the mechanisms of protein carbonylation, such as protein susceptibility and exact modification sites, but only for a limited number of proteins. Here we report the first proteome-wide study of carbonylated proteins including modification sites in HeLa cells for mild oxidative stress conditions. The analysis relied on our recent strategy utilizing mass spectrometry-based enrichment of carbonylated peptides after DNPH derivatization. Thus a total of 210 carbonylated proteins containing 643 carbonylation sites were consistently identified in three replicates. Most carbonylation sites (284, 44.2%) resulted from oxidation of lysine residues (aminoadipic semialdehyde). Additionally, 121 arginine (18.8%), 121 threonine (18.8%), and 117 proline residues (18.2%) were oxidized to reactive carbonyls. The sequence motifs were significantly enriched for lysine and arginine residues near carbonylation sites (±10 residues). Gene Ontology analysis revealed that 80% of the carbonylated proteins originated from organelles, 50% enrichment of which was demonstrated for the nucleus. Moreover, functional interactions between carbonylated proteins of kinetochore/spindle machinery and centrosome organization were significantly enriched. One-third of the 210 carbonylated proteins identified here are regulated during apoptosis.     

Characterization of a novel modification of a CHO-produced mAb: Evidence for the presence of tyrosine sulfation

Herein we describe the investigation of a Chinese hamster ovary (CHO)-expressed human mAb molecule found partially modified by a +80 Da adduct. This mass difference, suggestive of a single sulfation or phosphorylation addition, was observed by mass analysis of the intact and reduced molecule by mass spectrometry (MS). The modification was located on tyrosine 31 (Y31) of the light chain in the complementarity-determining region 1 by liquid chromatography (LC)-MS peptide mapping and electron transfer dissociation fragmentation. The complete loss of the 80 Da modification moiety during collision induced dissociation fragmentation suggested this modification could not be a tyrosine phosphorylation. Treatment of the mAb with alkaline phosphatase confirmed our hypothesis. Western blot experiment using anti-tyrosine sulfation antibody and LC retention time correlation with corresponding synthetic sulfated peptides further confirmed the identification of tyrosine sulfation on the light chain. The unique sequence motif with neighboring acidic amino acids and local secondary structure might play a role to make Y31 a substrate residue for sulfation. This type of modification, to our knowledge, has not been previously reported for CHO-produced human IgG antibodies.     

高质量抗体揭示Rad1蛋白在细胞中新的潜在活动机理

一组在进化上(从酵母到人)保守的基因Rad9、Rad1和Hus1在细胞周期监控点调控和DNA损伤修复中发挥重要作用．这三个蛋白可以形成环形异源三聚体,即9-1-1蛋白复合体．9-1-1复合体被认为是Rad9、Rad1和Hus1行使功能的主要形式．到目前为止,没有一个好的抗Rad1的抗体,严重阻碍了对Rad1和9-1-1复合体的研究．在本研究中,我们成功地制备了一株小鼠抗Rad1蛋白的单克隆抗体．这个抗体能够有效地检测小鼠和人的内源Rad1蛋白,可以用于酶联免疫吸附、蛋白质免疫印迹、免疫共沉淀和免疫荧光等实验．利用该抗体,我们发现在DNA损伤剂羟基脲(HU)的诱导下,小鼠Rad1蛋白在Rad9^+/+小鼠胚胎干细胞中表达明显增加,而在Rad9^-/-的小鼠胚胎干细胞中没有观察到该现象,这表明Rad9对Rad1的蛋白表达有调控作用．此外,内源的Rad1蛋白主要分布在细胞质中,在HU处理后并没有迁移进入细胞核的现象,这与先前广泛被人们所接受的在DNA损伤压力下Rad1和Hus1能够迁移进入细胞核并与Rad9形成9-1-1蛋白复合体的说法相矛盾．综合看来,Rad1和9-1-1蛋白复合体的分子作用机制比预期的要复杂,我们成功制备的Rad1单克隆抗体将成为研究Rad1以及9-1-1蛋白复合体的强有力的工具．     

Cooperative Binding of Stromal Interaction Molecule 1 (STIM1) to the N and C Termini of Calcium Release-activated Calcium Modulator 1 (Orai1)

Calcium flux through store-operated calcium entry is a central regulator of intracellular calcium signaling. The two key components of the store-operated calcium release-activated calcium channel are the Ca²⁺-sensing protein stromal interaction molecule 1 (STIM1) and the channel pore-forming protein Orai1. During store-operated calcium entry activation, calcium depletion from the endoplasmic reticulum triggers a series of conformational changes in STIM1 that unmask a minimal Orai1-activating domain (CRAC activation region (CAD)). To gate Orai1 channels, the exposed STIM1-activating domain binds to two sites in Orai1, one in the N terminus and one in the C terminus. Whether the two sites operate as distinct binding domains or cooperate in CAD binding is unknown. In this study, we show that the N and C-terminal domains of Orai1 synergistically contribute to the interaction with STIM1 and couple STIM1 binding with channel gating and modulation of ion selectivity.     

Fluorescent photoaffinity labeling of cytochrome P450 3A4 by lapachenole: identification of modification sites by mass spectrometry

While photoaffinity ligands (PALs) have been widely used to probe the structures of many receptors and transporters, their effective use in the study of membrane-bound cytochrome P450s is less established. Here, lapachenole has been used as an effective photoaffinity ligand of human P450 3A4, and mass spectrometry data demonstrating the efficient and specific photoaffinity labeling of CYP3A4 by this naturally occurring benzochromene compound is presented. Without photolysis, lapachenole is a substrate of CYP3A4 and can be metabolized to hydroxylated products by this enzyme. A high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) procedure was developed to analyze small amounts of intact purified CYP3A4, and analysis of the labeled protein showed the presence of one molecule of lapachenole bound per monomer of protein. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis after proteolytic digestion and isolation of fluorescent photolabeled peptides. Two peptide adducts accounting for >95% of the labeled peptides were isolated by HPLC, and both peptides, ECYSVFTNR (positions 97-105) and VLQNFSFKPCK (positions 459-469), were identified by nano-LC/ESI quadrupole time-of-flight (QTOF) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The sites of modification were further localized to positions Cys-98 and Cys-468 for each peptide by nano-LC/ESI QTOF tandem mass spectrometry (MS/MS). The results provided the first direct evidence for interaction between the PAL and the putative B-B' loop region, which may serve as a substrate access channel or as a part of the CYP3A4 active site. In conclusion, benzochromene analogues are effective PALs, which may be used in the study of other cytochrome P450 structures.     

组蛋白乙酰基转移酶1在肝癌细胞中介导的蛋白质乙酰化修饰谱分析

赖氨酸乙酰化是重要的蛋白质翻译后修饰之一,广泛存在于细胞的生理和病理过程.组蛋白乙酰基转移酶1(HAT1)作为第一个被鉴定的蛋白ε-氨基赖氨酸乙酰基转移酶,具有介导组蛋白和非组蛋白乙酰化的作用.然而,在肝癌细胞中HAT1介导的乙酰化蛋白质及其修饰位点目前仍不清楚.本研究首先揭示了 HAT1在肝癌组织中呈高表达,并且与预...     

Site-specific protein labeling with amine-containing molecules using Lactobacillus plantarum sortase

Modification of proteins with small molecules is a widely used and powerful tool in biological research. Enzymatic approaches are particularly promising because substrate specificity allows for site-specific modification. Sortase A, a transpeptidase from Staphylococcus aureus, cleaves between the T and G residues in the sequence LPXTG, and subsequently links the carboxyl group of the T residue to an amino group of N-terminal glycine oligomers by a native peptide bond. Although Gram-positive bacteria have several kinds of sortases, there are few reports concerning their expression and substrate specificity. Here, we demonstrate site-specific protein modification with primary amine-containing molecules catalyzed by Lactobacillus plantarum sortase. Enhanced green fluorescent protein (EGFP) was employed as a model protein, and an amine-containing biotin molecule was site-specifically conjugated with LPQTSEQ-tagged EGFP. We developed a novel Lactobacillus plantarum sortase that has different substrate specificity compared to Staphylococcus aureus sortase. Amine-directed protein modification was achieved using the Lactobacillus plantarum sortase 'LPQTSEQ' sequence original recognition tag. Our results demonstrate a promising method for expanding the capabilities of site-specific protein-small molecule modification.     

GABA metabolism and ROS induction in lentil (Lens culinaris Medik) plants by synthetic 1,2,3-Thiadiazole compounds

In this study, we investigated the physiological effects of three synthetic 1,2,3-thiadiazole compounds (compound I: 4-[1,2,3]-Thiadiazole-4-yl-phenol, compound II: 1,3-Bis[4-(1,2,3-thiadiazol-4-yl)phenoxy]propane, compound III: 4-(4-{2,3,4,5,6-Penta[4-(1,2,3-thiadiazole-4-yl)phenoxymethyl]benzyloxy}phenyl)-1,2,3-thiadiazole) treatments on plants via the characterization of γ-aminobutyric acid metabolite level, the accumulation of reactive oxygen species (ROS), total protein contents, total carbohydrate contents, fresh weight and dry mass in two lentil (Lens culinaris Medik) cultivars (Jordan 1 and Jordan 2). In response to the three synthetic 1,2,3-thiadiazole compounds (I, II and III) treatments. A significant increase in γ- aminobutyric acid (GABA) and malondialdehyde (MDA) levels and a significant reduction in carbohydrates and protein levels and fresh weight and dry mass were obtained in both lentil cultivars. Jordan 2 cultivar showed the highest GABA and MDA accumulation and significant reduction in the carbohydrate and protein levels under the various thiadiazole compounds treatments. This suggests that GABA molecule may act as a defense mechanism and signaling molecule in carbohydrate metabolism and other physiological systems in lentil. In conclusion, our results revealed that the synthetic 1,2,3-thiadiazole compound ?? (1,3-Bis[4-(1,2,3-thiadiazol-4-yl)phenoxy]propane) had the strongest physiological inhibitory effect on lentil.     

Rapid global characterization of immunoglobulin G1 following oxidative stress

ABSTRACT

Although peroxide and leachable metal-induced chemical modifications are among the most important quality attributes in bioprocess development, there is no mainstream characterization method covering all common modifications theoretically possible on therapeutic proteins that also gives consistent results quickly. Here, we describe a method for rapid and consistent global characterization of leachable metals- or peroxide-stressed immunoglobulin (Ig) G1 monoclonal antibodies (mAbs). Using two independent protease digestions, data-independent acquisition and data-dependent acquisition liquid chromatography high-resolution mass spectrometry, we monitored 55 potential chemical modifications on trastuzumab, a humanized IgG1 mAb. Processing templates including all observed peptides were developed on Skyline to consistently monitor all modifications throughout the stress conditions for both enzymatic digestions. The Global Characterization Data Processing Site, a universal automated data processing application, was created to batch process data, plot modification trends for peptides, generate sortable and downloadable modification tables, and produce Jmol code for three-dimensional structural models of the analyzed protein. In total, 53 sites on the mAb were found to be modified. Oxidation rates generally increased with the peroxide concentration, while leachable metals alone resulted in lower rates of modifications but more oxidative degradants. Multiple chemical modifications were found on IgG1 surfaces known to interact with Fc?RIII, complement protein C1q, and FcRn, potentially affecting activity. The combination of Skyline templates and the Global Characterization Data Processing Site results in a universally applicable assay allowing users to batch process numerous modifications. Applying this new method to stability studies will promote a broader and deeper understanding of stress modifications on therapeutic proteins.     

A method for specific chemical modification of gamma-carboxyglutamic acid residues in proteins

A method for the chemical modification of gamma-carboxyglutamic acid (Gla) residues in proteins is introduced that has the combined advantages of mildness, a high degree of specificity, and the ability to introduce a radiolabel at modification sites for ease in quantitation. Unlike other Gla modification procedures which are performed in the lyophilized state at 110 degrees C, this procedure is carried out in solution at 37 degrees C. The addition of morpholine and formaldehyde to a slightly acidic solution of bovine prothrombin fragment 1 (residues 1-156) results in the conversion of Gla residues to gamma- methyleneglutamic acid (gamma- MGlu ). The extent of modification is controlled by the relative amounts of modification reagents to protein. A 100-fold molar excess of reagents to fragment 1 produced a protein molecule containing two gamma- MGlu residues, while a modification run at 10,000-fold molar excess of reagents to protein yielded fragment 1 containing eight gamma- MGlu residues per molecule. The specificity of this modification is illustrated by the interaction of native and modified protein with antibody populations directed against fragment 1. Native fragment 1, 8 gamma- MGlu fragment 1, and 2 gamma- MGlu fragment 1 show fairly similar behavior toward whole anti-fragment 1 serum. Differential behavior was exhibited by the native and modified proteins toward a subpopulation of antibodies specific to the calcium ion conformation of fragment 1. Unmodified fragment 1 displayed a strong affinity for these antibodies; however, the 2 gamma- MGlu fragment 1 exhibited a moderate affinity and the 8 gamma- MGlu fragment 1 did not bind to these antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional protein microarray: an ideal platform for investigating protein binding property

Functional protein microarray is an important tool for high-throughput and large-scale systems biology studies.Besides the progresses that have been made for protein microarray fabrication,significant ...     

TRIF Modulates TLR5-dependent Responses by Inducing Proteolytic Degradation of TLR5

Proteolytic modification of pattern recognition receptors and their signaling adaptor molecules has recently emerged as an essential cellular event to regulate immune and inflammatory responses. Here we show that the TIR domain containing adaptor-inducing interferon-β (TRIF), an adaptor molecule mediating TLR3 signaling and MyD88-independent signaling of TLR4, plays an inhibitory role in TLR5-elicited responses by inducing proteolytic degradation of TLR5. TRIF overexpression in human embryonic kidney (HEK293) and human colonic epithelial (NCM460) cells abolishes the cellular protein level of TLR5, whereas it does not alter TLR5 mRNA level. Thus, TRIF overexpression dramatically suppresses flagellin/TLR5-deriven NFκB activation in NCM460 cells. TRIF-induced TLR5 protein degradation is completely inhibited in the presence of pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone), whereas several specific inhibitors against cathepsin B, reactive oxygen species, or ubiquitin-mediated proteasome activity fail to suppress this degradation. These results indicate that TRIF-induced caspase activity causes TLR5 protein degradation. In addition, we identify that the C terminus of TRIF and extracellular domain of TLR5 are required for TRIF-induced TLR5 degradation. Furthermore, TRIF-induced proteolytic degradation is extended to TLR3, TLR6, TLR7, TLR8, TLR9, and TLR10, whereas the cellular level of TLR1, TLR2, and TLR4 is not affected by TRIF overexpression. These results suggest that, in addition to mediating TLR3- or TLR4-induced signaling as an adaptor molecule, TRIF can participate in proteolytic modification of certain members of TLRs to modulate the functionality of TLRs at post-translational level. Collectively, our findings propose a potential inhibitory role of TRIF at least in regulating host-microbial communication via TLR5 in colonic epithelial cells.     

Divergent pathways for the angiotensin-(1-12) metabolism in the rat circulation and kidney

Evidence of endogenous angiotensin-(1-12) [Ang-(1-12)] may necessitate revision of the accepted view that Ang I is the immediate peptide product derived from the precursor protein angiotensinogen. As the processing of this peptide has not been fully elucidated, we characterized Ang-(1-12) metabolism in the serum and kidney of the mRen2.Lewis rat, a model of high circulating renin and ACE expression. A sensitive HPLC-based method to detect the metabolism ex vivo of low concentrations of (125)I-labeled Ang-(1-12) was utilized. Ang-(1-12) processing to serum did not reveal the participation of renin; however, serum ACE readily converted Ang-(1-12) to Ang I with subsequent metabolism to Ang II. Ang I and Ang II forming activities for serum ACE were 102±4 and 104±3 fmol/ml/min serum (n=3), respectively, and both products were abolished by the potent ACE inhibitor lisinopril. The metabolism of Ang-(1-12) in renal cortical membranes also revealed the formation of Ang I; however, the main products were Ang-(1-7) and Ang-(1-4) at 129±9 and 310±12 fmol/mg/min protein (n=4), respectively. Neprilysin inhibition abolished these products and substantially reduced the overall metabolism of Ang-(1-12). Incubation of Ang-(1-12) with either human or mouse neprilysin revealed identical products. We conclude that endogenous Ang-(1-12) may contribute to the expression of biologically active angiotensins through a renin-independent pathway. The preferred route for Ang-(1-12) metabolism likely reflects the relative tissue content of ACE and neprilysin.