Prevalent Post-translational Modification Research Articles

Unraveling the Complexity of Ubiquitin Signaling

Protein ubiquitination, the covalent attachment of ubiquitin to target proteins, has emerged as one of the most prevalent posttranslational modifications (PTMs), regulating nearly every cellular pathway. The diversity of signaling associated with this particular PTM stems from the myriad ways in which a target protein can be modified by ubiquitin, e.g., monoubiquitin, multi-monoubiquitin, and polyubiquitin linkages. In this Review, we focus on developments in both enzymatic and chemical methods that engender ubiquitin with new chemical and physical properties. Moreover, we highlight how these methods have enabled studies directed toward (i) characterizing enzymes responsible for reversing the ubiquitin modification, (ii) understanding the influence of ubiquitin on protein function and crosstalk with other PTMs, and (iii) uncovering the impact of polyubiquitin chain linkage and length on downstream signaling events.

Mapping ubiquitin modifications reveals new functions for the yeast nuclear pore complex

Covalent attachment of ubiquitin to target proteins, or ubiquitylation, has emerged as one of the most prevalent posttranslational modifications (PTMs), regulating nearly every cellular pathway. The diversity of functions associated with this particular PTM stems from the myriad ways in which a target protein can be modified by ubiquitin, e.g., monoubiquitin, multi-monoubiquitin and polyubiquitin linkages. In the current study, we took a systematic approach to analyze the ubiquitylation profiles of the yeast Saccharomyces cerevisiae nuclear pore complex (NPC) proteins or nucleoporins. We found the yeast NPC to be extensively modified by ubiquitin with highly variable ubiquitylation profiles, suggesting that dissection of these modifications may provide new insights into the regulation of NPC functions and reveal additional roles for nucleoporins beyond nuclear transport.

Glutathionylation at Cys-111 Induces Dissociation of Wild Type and FALS Mutant SOD1 Dimers

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1(WT) dimers, increasing the equilibrium dissociation constant K(d) to ~10-20 μM, comparable to that of the aggressive A4V mutant. SOD1(A4V) is further destabilized by glutathionylation, experiencing an ~30-fold increase in K(d). Dissociation kinetics of glutathionylated SOD1(WT) and SOD1(A4V) are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1(I112T) has a modestly increased dissociation rate but no change in K(d) when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.

The aberrance of the 4S diastereomer of 4-hydroxyproline.

Prolyl 4-hydroxylases install a hydroxyl group in the 4R configuration on the gamma-carbon atom of certain (2S)-proline (Pro) residues in tropocollagen, elastin, and other proteins to form (2S,4R)-4-hydroxyproline (Hyp). The gauche effect arising from this prevalent post-translational modification enforces a C(gamma)-exo ring pucker and stabilizes the collagen triple helix. The Hyp diastereomer (2S,4S)-4-hydroxyproline (hyp) has not been observed in a protein, despite the ability of electronegative 4S substituents to enforce the more common C(gamma)-endo ring pucker of Pro. Here, we use density functional theory, spectroscopy, crystallography, and calorimetry to explore the consequences of hyp incorporation on protein stability using a collagen model system. We find that the 4S-hydroxylation of Pro to form hyp does indeed enforce a C(gamma)-endo ring pucker but a transannular hydrogen bond between the hydroxyl moiety and the carbonyl of hyp distorts the main-chain torsion angles that typically accompany a C(gamma)-endo ring pucker. This same transannular hydrogen bond enhances an n-->pi* interaction that stabilizes the trans conformation of the peptide bond preceding hyp, endowing hyp with the unusual combination of a C(gamma)-endo ring pucker and high trans/cis ratio. O-Methylation of hyp to form (2S,4S)-4-methoxyproline (mop) eliminates the transannular hydrogen bond and restores a prototypical C(gamma)-endo pucker. mop residues endow the collagen triple helix with much more conformational stability than do hyp residues. These findings highlight the critical importance of the configuration of the hydroxyl group installed on C(gamma) of proline residues.

Site-specific Phosphorylation of CXCR4 Is Dynamically Regulated by Multiple Kinases and Results in Differential Modulation of CXCR4 Signaling

The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.

Strategies for the identification of kinase substrates using analog-sensitive kinases

Phosphorylation of proteins is a prevalent post-translational modification, which affects intracellular signaling in many ways. About 2% of all eukaryotic genes code for protein kinases catalyzing phosphorylation events. Despite technological advances that have made it possible to identify thousands of phosphorylation sites simultaneously, identification of the substrates of a given kinase remains an exceptionally challenging task. Here, we summarize approaches for substrate identification that make use of genetically engineered ‘analog-sensitive’ kinases.

Cooperativity within proximal phosphorylation sites is revealed from large-scale proteomics data

BackgroundPhosphorylation is the most prevalent post-translational modification on eukaryotic proteins. Multisite phosphorylation enables a specific combination of phosphosites to determine the speed, specificity and duration of biological response. Until recent years, the lack of high quality data limited the possibility for analyzing the properties of phosphorylation at the proteome scale and in the context of a wide range of conditions. Thanks to advances of mass spectrometry technologies, thousands of phosphosites from in-vivo experiments were identified and archived in the public domain. Such resource is appropriate to derive an unbiased view on the phosphosites properties in eukaryotes and on their functional relevance.ResultsWe present statistically rigorous tests on the spatial and functional properties of a collection of ~70,000 reported phosphosites. We show that the distribution of phosphosites positioning along the protein tends to occur as dense clusters of Serine/Threonines (pS/pT) and between Serine/Threonines and Tyrosines, but generally not as much between Tyrosines (pY) only. This phenomenon is more ubiquitous than anticipated and is pertinent for most eukaryotic proteins: for proteins with ≥ 2 phosphosites, 54% of all pS/pT sites are within 4 amino acids of another site. We found a strong tendency for clustered pS/pT to be activated by the same kinase. Large-scale analyses of phosphopeptides are thus consistent with a cooperative function within the cluster.ConclusionsWe present evidence supporting the notion that clusters of pS/pT but generally not pY should be considered as the elementary building blocks in phosphorylation regulation. Indeed, closely positioned sites tend to be activated by the same kinase, a signal that overrides the tendency of a protein to be activated by a single or only few kinases. Within these clusters, coordination and positional dependency is evident. We postulate that cellular regulation takes advantage of such design. Specifically, phosphosite clusters may increase the robustness of the effectiveness of phosphorylation-dependent response.ReviewersReviewed by Joel Bader, Frank Eisenhaber, Emmanuel Levy (nominated by Sarah Teichmann). For the full reviews, please go to the Reviewers' comments section.

Completely Different Effects of Desferrioxamine on Hemin/Nitrite/H2O2-Induced Bovine Serum Albumin Nitration and Oxidation

Protein tyrosine nitration is becoming increasingly recognized as a prevalent post-translational modification that could serve as a biomarker of nitric oxide (NO)-mediated oxidative stress. One received protein nitration model is the heme- or hemoprotein-dependent pathway that involves the contribution of iron and the formation of free radicals. Therefore, the iron chelating agent desferrioxamine (DFO) can affect the development of oxidative and nitrative stress. In the hemin/nitrite/H2O2 system-induced bovine serum albumin (BSA) nitration and oxidation model, BSA was analyzed for 3-nitrotyosine and carbonyl groups measured by spectrophotometry, SDS-PAGE, and Western blotting upon exposure to DFO. The results showed a significant dose-dependent inhibitory effect of DFO on BSA nitration, while an enhancement on oxidation was surprisingly observed. The promotion on protein oxidation could not be the result of the formation of ferrioxamine since the antinitration and prooxidant effect of DFO was abolished when it combined with Fe3+ to form ferrioxamine. Our studies also indicated that the abnormal effect of DFO on promoting protein oxidation probably originated from the hemin-DFO complex, which needs further study. The completely different effects of DFO on hemin-induced protein tyrosine nitration and protein oxidation should be taken into account when DFO is used in experimental and clinical applications.

Nano titanium dioxide photocatalytic protein tyrosine nitration: A potential hazard of TiO 2 on skin

Protein tyrosine nitration is a prevalent post-translational modification which occurs as a result of oxidative and nitrative stress, it may be directly involved in the onset and/or progression of diseases. Considering the existence of nano titanium dioxide (TiO 2) in environment and sunscreen products along with the high content of nitrite in sweat, the UV-exposed skin may be a significant target for the photosensitized damage. In this paper, tyrosine nitration of bovine serum albumin (BSA) was initiated in the UV-irradiated reaction mixture containing 0.2–3.0 mg/ml of three commercially nano TiO 2 products and 0.25–1.0 mM NO 2 - . It was found that anatase TiO 2 and Degussa P25 TiO 2 showed prominent photocatalytic activity on promoting the formation of protein tyrosine nitration, and the optimum condition for the reaction was around physiological pH. Meanwhile, the photocatalytic effect of rutile on protein tyrosine nitration was subtle. The potential physiological significance of nano TiO 2-photocatalytic protein nitration was also demonstrated in mouse skin homogenate. Although the relationship between photocatalytic protein tyrosine nitration and chronic cutaneous diseases needs further study, the toxicity of nano TiO 2 to the skin disease should be paid more attention in the production and utilization process.

Arginine methylation at a glance

Arginine methylation is a prevalent post-translational modification found on both nuclear and cytoplasmic proteins. The methylation of arginine residues is catalyzed by the protein arginine N -methyltransferase (PRMT) family of enzymes. Proteins that are arginine methylated are involved in a number

S-nitrosylation: NO-related redox signaling to protect against oxidative stress.

Nitric oxide (NO) plays an important role in the regulation of cardiovascular function. S-nitrosylation, the covalent attachment of an NO moiety to sulfhydryl residues of proteins, resulting in the formation of S-nitrosothiols (SNOs), is a prevalent posttranslational protein modification involved in redox-based cellular signaling. Under physiologic conditions, protein S-nitrosylation and SNOs provide protection preventing further cellular oxidative and nitrosative stress. However, oxidative stress and the resultant dysfunction of NO signaling have been implicated in the pathogenesis of cardiovascular diseases.

Hydration of a glycoprotein: relative water affinity of peptide and glycan moieties

Glycosylation, the most prevalent post-translational modification of proteins, affects a number of physical properties including the interactions with the surrounding aqueous solvent. Such glycan-water interactions have been discussed with respect to the increased solubility generally observed for glycoproteins, but experimental support of this correlation remains sparse. We have applied a two-channel calorimetric method to measure the free energy and enthalpy of hydration at 25 degrees C for the glycoprotein phytase (Phy) and a deglycosylated form (dgPhy) of the same protein. Comparisons of results for Phy and dgPhy show that the polypeptide moiety has a higher affinity for water than the glycans. In fact, at moderate hydration levels (approximately 0.3 g water/g macromolecule) the water uptake appears to be entirely governed by adsorption to the peptide groups. We conclude that strengthened interaction with the solvent is unlikely to be the mechanism underlying the increased solubility and lowered propensity of aggregation often reported to result from the glycosylation of proteins.

Poly-ADP-ribosylation is a prevalent post-translational modification of mitochondrial proteins in the brain

Poly-ADP-ribosylation is a prevalent post-translational modification of mitochondrial proteins in the brain

Fragmentation pathways of N G-methylated and unmodified arginine residues in peptides studied by ESI-MS/MS and MALDI-MS

Protein methylation at arginine residues is a prevalent posttranslational modification in eukaryotic cells that has been implicated in processes from RNA-binding and transporting to protein sorting and transcription activation. Three main forms of methylarginine have been identified: N G-monomethylarginine (MMA), asymmetric N G,N G-dimethylarginine (aDMA), and symmetric N G,N′ G-dimethylarginine (sDMA). To investigate gas-phase fragmentations and characteristic ions arising from methylated and unmodified arginine residues in detail, we subjected peptides containing these residues to electrospray triple-quadrupole tandem mass spectrometry. A variety of low mass ions including (methylated) ammonium, carbodiimidium, and guanidinium ions were observed. Fragment ions resulting from the loss of the corresponding neutral fragments (amines, carbodiimide, and guanidine) from intact molecular ions as well as from N- and C-terminal fragment ions were also identified. Furthermore, the peptides containing either methylated or unmodified arginines gave rise to abundant fragment ions at m/ z 70, 112, and 115, for which cyclic ion structures are proposed. Electrospray ionization tandem mass spectra revealed that dimethylammonium ( m/ z 46) is a specific marker ion for aDMA. A precursor ion scanning method utilizing this fragment ion was developed, which allowed sensitive and specific detection of aDMA-containing peptides even in the presence of a five-fold excess of phosphorylase B digest. Interestingly, regular matrix-assisted laser desorption/ionization mass spectra recorded from aDMA- or sDMA-containing peptides showed metastable fragment ions resulting from cleavages of the arginine side chains. The neutral losses of mono- and dimethylamines permit the differentiation between aDMA and sDMA.

The Novel Human Protein Arginine N-Methyltransferase PRMT6 Is a Nuclear Enzyme Displaying Unique Substrate Specificity

Protein arginine methylation is a prevalent posttranslational modification in eukaryotic cells that has been implicated in signal transduction, the metabolism of nascent pre-RNA, and the transcriptional activation processes. In searching the human genome for protein arginine N-methyltransferase (PRMT) family members, a novel gene has been found on chromosome 1 that encodes for an apparent methyltransferase, PRMT6. The polypeptide chain of PRMT6 is 41.9 kDa consisting of a catalytic core sequence common to other PRMT enzymes. Expressed as a glutathione S-transferase fusion protein, PRMT6 demonstrates type I PRMT activity, capable of forming both omega-N(G)-monomethylarginine and asymmetric omega-N(G),N(G)-dimethylarginine derivatives on the recombinant glycine- and arginine-rich substrate in a processive manner with a specific activity of 144 pmol methyl groups transferred min(-1) mg(-1) enzyme. A comparison of substrate specificity reveals that PRMT6 is functionally distinct from two previously characterized type I enzymes, PRMT1 and PRMT4. In addition, PRMT6 displays automethylation activity; it is the first PRMT to do so. This novel human PRMT, which resides solely in the nucleus when fused to the green fluorescent protein, joins a family of enzymes with diverse functions within cells.

Peptide amidating enzymes are present in cultured endothelial cells

Car☐y-terminal amidation is a prevalent post-translational modification necessary for the bioactivity of many peptides. We now report that the two enzymes essential for amidation, peptidylglycine α-monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL), are present in both the cytosol and membrane fractions of cultured bovine aortic endothelial cells. Endothelial PAM exhibits ascorbate-dependent turnover and is inactivated by the mechanism-based inactivator, 4-phenyl-3-butenoic acid (PBA), whereas PGL activity is independent of ascorbate and is not affected by PBA. These enzymological characteristics correspond to those of amidating enzymes from other tissues. These results suggest a heretofore unrecognized role for α-amidated peptides in cardiovascular function.

A new facile trinitrophenylated substrate for peptide α-amidation and its use to characterize PAM activity in chromaffin granules

Carboxyl terminal α-amidation is a prevalent post translational modification in neuropeptide hormones, with amidation being essential for biological activity. We report a direct demonstration and characterization of peptidyl α-amidating monooxygenase (PAM) activity in chromaffin granules, secretory vesicles long known as loci for synthesis and storage of catecholamines but only recently recognized as processing and storage sites for neuropeptides. This finding, together with the recently recognized competence of dopamine-b-monooxygenase to carry out N-dealkylation, provides important information regarding the co-localization and co-secretion of multiple neuromodulators. In addition, we introduce a new substrate for both pituitary and chromaffin granule PAM—TNP-D-Tyr-Val-Gly. This substrate exhibits high turnover, and has the important advantage of allowing quantitative activity determinations using standard spectrophotometric techniques, thus facilitating mechanistic studies and inhibitor development.