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PubMed1994

Oxidation of low-density lipoprotein by hypochlorite causes aggregation that is mediated by modification of lysine residues rather than lipid oxidation.

Peroxidation of low-density lipoprotein (LDL) lipid is generally thought to represent the initial step in a series of modification reactions that ultimately transform the protein moiety of the lipoprotein into a form recognized by receptors different from those that bind native LDL. Uptake of LDL via these alternative receptors can lead to the formation of lipid-laden cells, which are typical for the early stages of atherogenesis. We have studied the oxidative modification of LDL by hypochlorite (-OCl), a powerful oxidant produced from H2O2 and chloride via the action of myeloperoxidase which is released from activated neutrophils and monocytes. Exposure of LDL to reagent or enzymically generated -OCl at 4 or 37 degrees C resulted in immediate and preferential oxidation of amino acid residues of apolipoprotein B-100, the single protein associated with LDL. Lysine residues quantitatively represented the major target and, like tryptophan, were oxidized to approximately the same extent with reagent or enzymically generated -OCl. In contrast, LDL lipid oxidation was less favoured than protein oxidation, as judged by the amounts of lipid hydroperoxides, chlorohydrins, cholesterol or fatty acid oxidation products formed. Treatment with -OCl caused aggregation of LDL, as shown by an increased turbidity of the oxidized LDL solution and elution from a size-exclusion h.p.l.c. column of high-molecular-mass LDL complexes. Chemical modification of lysine residues before oxidation with -OCl prevented aggregation, while it enhanced the extent of lipid peroxidation. Treatment of LDL with -OCl also caused the formation of carbonyl groups and release of ammonia; both these modifications were inhibited by lysine-residue modification before oxidation. These results demonstrate that aggregation reactions are dependent on initial lysine oxidation by -OCl, followed by deamination and carbonyl formation, but do not involve lipid (per)oxidation. We propose that the observed -OCl-mediated aggregation of LDL is caused, at least in part, by cross-linking of apoproteins by Schiff-base formation independently of lipid peroxidation.

Adult↗
PubMed1995

Chemical modification and inactivation of phospholipases A2 by a manoalide analogue.

Chemical modification and inactivation of bovine pancreatic, porcine pancreatic, Naja naja atra and Pseudechis australis phospholipases A2 (PLA2s), belonging to Group I, and of Trimeresurus flavoviridis, Vipera russelli russelli and Agkistrodon halys blomhoffii PLA2s, belonging to Group II, were investigated by the use of a manoalide (MLD)-analogue, 1-(2,5-dihydro-hydroxy-5-oxo-3-furanyl)-8,12-dimethyl-4-formyl-3,7, 11-tridecatrienol. At appropriate time intervals, residual PLA2 activities towards monodispersed, anionic mixed micellar and non-ionic mixed micellar substrates were measured. We tested the protective effect of micellar n-dodecylphosphocholine (n-C12PC) on enzyme inactivation. Inactivation of pancreatic PLA2s (Group I) was only observed towards anionic mixed micellar substrates. This inactivation was completely prevented by the presence of micellar n-C12PC. From a fragmentation study of modified bovine pancreatic PLA2 using lysyl endopeptidase, we speculated that Lys-56 of this enzyme was modified by MLD-analogue and that this modification was responsible for enzyme inactivation. Inactivation of non-pancreatic PLA2s was observed towards all types of substrate, except that no significant inactivation of N. naja atra PLA2 (Group I) towards monodispersed substrate was noted. Micellar n-C12PC protected N. naja atra PLA2 (Group I) completely from inactivation by MLD-analogue, but had lesser protective effects on P. australis PLA2 (Group I), T. flavoviridis and V. russelli russelli PLA2s (Group II). However, no significant protection of A. halys blomhoffii PLA2s (Group II) activity was observed. These results indicate that the inactivation of pancreatic and N. naja atra PLA2s originates from the modification of Lys residues at the interfacial recognition site, and that inactivation of P. australis, T. flavoviridis and V. russelli PLA2s arises from the modification of Lys residues at the catalytic site, interfacial recognition site and regions outside both sites. The inactivation of A. halys blomhoffii PLA2 was assumed to be due to the modification of Lys residues outside the two sites described above.

Amino Acid Sequence↗
PubMed1998

Activation mechanism and modification kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate.

Substrate effects on the activation kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate (pCMB) have been studied. On the basis of the kinetic equation of substrate reaction in the presence of pCMB, all modification kinetic constants for the free enzyme and enzyme-substrate binary and ternary complexes have been determined. The results of the present study indicate that the modification of Chinese hamster dihydrofolate reductase by pCMB shows single-phase kinetics, and that changes in the enzyme activity and tertiary structure proceed simultaneously during the modification process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against modification by pCMB. In the presence of a saturating concentration of NADPH, the value of kcat for 7,8-dihydrofolate in the enzyme-catalysed reaction increased four-fold on modification of Cys-6, accompanied by a two-fold increase in Km for the modified enzyme. The utilization of the binding energy of a group to increase kcat rather than reduce Km implies that the full binding energy of the group is not realized in the formation of the enzyme-substrate complex, but is used to stabilize the enzyme-transition-state complex.

Animals↗
PubMed2001

Experimental genome evolution: large-scale genome rearrangements associated with resistance to replacement of a chromosomal restriction-modification gene complex.

Type II restriction enzymes are paired with modification enzymes that protect type II restriction sites from cleavage by methylating them. A plasmid carrying a type II restriction-modification gene complex is not easily replaced by an incompatible plasmid because loss of the former leads to cell death through chromosome cleavage. In the present work, we looked to see whether a chromosomally located restriction-modification gene complex could be replaced by a homologous stretch of DNA. We tried to replace the PaeR7I gene complex on the Escherichia coli chromosome by transducing a homologous stretch of PaeR7I-modified DNA. The replacement efficiency of the restriction-modification complex was lower than expected. Some of the resulting recombinant clones retained the recipient restriction-modification gene complex as well as the homologous DNA (donor allele), and slowly lost the donor allele in the absence of selection. Analysis of their genome-wide rearrangements by Southern hybridization, inverse polymerase chain reaction (iPCR) and sequence determination demonstrated the occurrence of unequal homologous recombination between copies of the transposon IS3. It was strongly suggested that multiple rounds of unequal IS3-IS3 recombination caused large-scale duplication and inversion of the chromosome, and that only one of the duplicated copies of the recipient PaeR7I was replaced.

Chromosomes, Bacterial↗
PubMed2002

Structural heterogeneity of carbohydrate modifications affects serospecificity of Campylobacter flagellins.

Flagellin from Campylobacter coli VC167 is post-translationally modified at > or = 16 amino acid residues with pseudaminic acid and three related derivatives. The predominant modification was 5,7-diacetamido-3,5,7,9 - tetradeoxy - l - glycero - l - manno - nonulosonic acid (pseudaminic acid, Pse5Ac7Ac), a modification that has been described previously on flagellin from Campylobacter jejuni 81-176. VC167 lacked two modi-fications present in 81-176 and instead had two unique modifications of masses 431 and 432 Da. Flagellins from both C. jejuni 81-176 and C. coli VC167 were also modified with an acetamidino form of pseudaminic acid (PseAm), but tandem mass spectrometry indicated that the structure of PseAm differed in the two strains. Synthesis of PseAm in C. coli VC167 requires a minimum of six ptm genes. In contrast, PseAm is synthesized in C. jejuni 81-176 via an alternative pathway using the product of the pseA gene. Mutation of the ptm genes in C. coli VC167 can be detected by changes in apparent Mr of flagellin in SDS-PAGE gels, changes in isoelectric focusing (IEF) patterns and loss of immunoreactivity with antiserum LAH2. These changes corresponded to loss of both 315 Da and 431 Da modifications from flagellin. Complementation of the VC167 ptm mutants with the 81-176 pseA gene in trans resulted in flagellins containing both 315 and 431 Da modifications, but these flagellins remained unreactive in LAH2 antibody, suggesting that the unique form of PseAm encoded by the ptm genes contributes to the serospecificity of the flagellar filament.

Amino Acid Sequence↗
PubMed2000

Follow-up of melanocytic skin lesions with digital epiluminescence microscopy: patterns of modifications observed in early melanoma, atypical nevi, and common nevi.

BACKGROUND: Digital epiluminescence microscopy (DELM) has been reported to be a useful technique for the follow-up of melanocytic nevi. One of the promises of this technique is to identify modifications over time that indicate impending or incipient malignancy and to facilitate surveillance of melanocytic skin lesions, particularly in patients with multiple clinically atypical nevi. OBJECTIVE: Our purpose was to report on patterns of modifications over time observed in benign melanocytic skin lesions and melanoma. METHODS: A total of 1862 sequentially recorded DELM images of melanocytic lesions from 202 patients (mean age, 36.1 years; 54.0% female patients) with multiple clinically atypical nevi were included in the analysis. The median follow-up interval was 12. 6 months. Melanocytic lesions with substantial modifications over time (enlargement, changes in shape, regression, color changes or appearance of ELM structures known to be associated with melanoma) were excised and referred to histopathologic examination. RESULTS: A total of 75 melanocytic skin lesions (4.0%) from 52 patients (mean age, 33.3 years; 63.5% female patients) showed substantial modifications over time and were excised and referred to histopathologic examination. Eight changing lesions were histologically diagnosed as early melanomas. These lesions frequently showed focal enlargement associated with a change in shape as well as appearance of ELM structures that are known to be associated with melanoma. In contrast, the majority of benign changing lesions (common and atypical nevi) showed symmetric enlargement without substantial structural ELM changes. Six of the 8 patients in whom melanoma developed were unaware of the fact that the lesion had changed over time. CONCLUSION: We demonstrate that follow-up of melanocytic lesions with DELM helps to identify patterns of morphologic modifications typical for early melanoma. DELM may therefore serve as a useful tool to improve the surveillance of patients with multiple atypical nevi.

Adult↗
PubMed2005

Extensive 3' modification of plant small RNAs is modulated by helper component-proteinase expression.

RNA silencing is an evolutionarily conserved process in eukaryotes that represses gene expression by using 21- to 24-nt guide RNAs to mediate mRNA cleavage or translational inhibition. Plants have two distinct groups of silencing-associated small RNAs (smRNAs): the micro RNAs (miRNAs) and the small interfering RNAs (siRNAs). A recent report by Yu et al. [Yu, B., Yang, Z., Li, J., Minakhina, S., Yang, M., Padgett, R. W., Steward, R. & Chen, X. (2005) Science 307, 932-935] has shown that plant miRNAs are modified at their 3' termini with a methyl group. Here, we show that a large fraction of all silencing-associated smRNAs in tobacco are modified; this modification occurs on the 2' hydroxyl of the terminal ribose and significantly reduces the cloning efficiency of these modified smRNAs. Expression of the strong silencing suppressor P1/helper-component proteinase results in a marked decrease in the 3'-terminal modification of viral siRNAs but does not significantly affect the modification of endogenous miRNAs and 24-nt siRNAs. The differential modification mediated by helper-component proteinase expression implies that exogenous and endogenous smRNAs are processed through independent pathways that are isolated by subcellular compartmentalization and/or the association with distinct Dicer complexes. The degree of terminal modification may play an important role in regulating the extent to which primary smRNA signals can be amplified by RNA-dependent RNA polymerases.

Gene Expression Regulation, Plant↗
PubMed2002

Posttranslational modifications in the CP43 subunit of photosystem II.

Photosystem II (PSII) catalyzes the light-driven oxidation of water and the reduction of plastoquinone; the oxidation of water occurs at a cluster of four manganese. The PSII CP43 subunit functions in light harvesting, and mutations in the fifth luminal loop (E) of CP43 have established its importance in PSII structure and/or assembly [Kuhn, M. G. & Vermaas, V. F. J. (1993) Plant Mol. Biol. 23, 123-133]. The sequence A(350)PWLEPLR(357) in luminal loop E is conserved in CP43 genes from 50 organisms. To map important posttranslational modifications in this sequence, tandem mass spectrometry (MS/MS) was used. These data show that the indole side chain of Trp-352 is posttranslationally modified to give mass shifts of +4, +16, and +18 daltons. The masses of the modifications suggest that the tryptophan is modified to kynurenine (+4), a keto-/amino-/hydroxy- (+16) derivative, and a dihydro-hydroxy- (+18) derivative of the indole side chain. Peptide synthesis and MS/MS confirmed the kynurenine assignment. The +16 and +18 tryptophan modifications may be intermediates formed during the oxidative cleavage of the indole ring to give kynurenine. The site-directed mutations, W352C, W352L, and W352A, exhibit an increased rate of photoinhibition relative to wild type. We hypothesize that Trp-352 oxidative modifications are a byproduct of PSII water-splitting or electron transfer reactions and that these modifications target PSII for turnover. As a step toward understanding the tertiary structure of this CP43 peptide, structural modeling was performed by using molecular dynamics.

Algorithms↗
PubMed1992

Protein surface topology-probing by selective chemical modification and mass spectrometric peptide mapping.

Aminoacetylation of lysine residues and the modification of arginine by 1,2-cyclohexanedione to N7,N8-(dihydroxy-1,2-cyclohexylidene)arginine were used for probing the surface topology of hen-eggwhite lysozyme as a model protein. The molecular identification of lysine and arginine modification sites was provided by molecular weight determinations of modified and unmodified tryptic peptide mixtures (peptide mapping) using 252Cf plasma desorption mass spectrometry. At conditions of limited chemical modification, mass-spectrometric peptide-mapping analyses of lysozyme derivatives enabled the direct assignment of relative reactivities of lysine and arginine residues at different reaction times and reagent concentrations. The relative reactivities of lysine residues showed a direct correlation with their surface accessibilities from x-ray structure data. For the reaction with 1,2-cyclohexanedione, a selective modification at Arg-5, -125, -112, and -73 was identified, and an inverse correlation of relative reactivities with the surface accessibility ratios of the N7- and the N8-guanidino functions was obtained. By examination of the x-ray structural data of lysozyme, this selective modification was attributed to intramolecular catalysis because of the presence of neighboring proton acceptor groups, such as the Asp-119 carboxylate group for Arg-125 and the Trp-123 and Arg-125 carbonyl groups for Arg-5.

Acetylation↗
PubMed1996

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.

Base Sequence↗
PubMed1995

Protein modification by ADP-ribose via acid-labile linkages.

As substrate for protein-mono-ADP-ribosyltransferases, NAD has been shown to be the donor of ADP-ribose to many different nucleophiles found in proteins. This post-translational modification of proteins has been implicated in the regulation of membrane-associated processes including signal transduction, muscle cell differentiation, and protein trafficking and secretion. Described here is the preparation and chemical characterization of low molecular weight conjugates that were used as models for an acetal linkage between ADP-ribose and the hydroxyl group of a protein acceptor such as serine, threonine, tyrosine, hydroxyproline, or hydroxylysine residues. Model conjugates of ADP-ribose containing an acetal linkage were prepared, their structures were established by NMR, and the chemical stability of the linkage to ADP-ribose was studied and compared to the other known ADP-ribosyl-amino acid linkages. The rapid release of intact ADP-ribose from the acetal model conjugates in 44% formic acid distinguished them chemically from all the other known ADP-ribosyl-amino acid modifications. Rat liver proteins were shown to be modified by ADP-ribose in vivo by acid-labile linkages, providing evidence for a new class of endogenous ADP-ribose modification of animal cell proteins. The amount of modification was approximately 16 pmol of ADP-ribose per mg of total protein, and proteins modified by acid-labile linkages were detected in all subcellular fractions examined, suggesting that the scope of this modification in vivo is broad.

Adenosine Diphosphate Ribose↗
PubMed1996

Keratan sulfate modification of CD44 modulates adhesion to hyaluronate.

CD44 alternative splicing has been implicated in the regulation of CD44 function. CD44 undergoes significant posttranslational modification in all cells, but the functional consequences of these modifications are poorly understood. In the current study, we have demonstrated that keratan sulfate modification of CD44 significantly modulates its ability to bind to hyaluronate. We observed naturally occurring differences in CD44 keratan sulfate substitution between two clonal variants of the KM12 human colon carcinoma cell line. CD44 on the highly metastatic KM12L4 clone is more heavily substituted with keratan sulfate than CD44 on the poorly metastatic KM12C6 clone. Moreover, CD44H on KM12L4 bound to hyaluronate poorly compared to CD44H on KM12C6. Removal of keratan sulfate from CD44 greatly enhanced CD44-mediated cell adhesion to hyaluronate. Removal of keratan sulfate from CD44H-immunoglobulin fusion proteins also enhanced their adhesion to hyaluronate. The influence of glycosaminoglycan substitution on CD44 function was specific to keratan sulfate substitution; treatment to remove chondroitin sulfate, heparan sulfate, or hyaluronate did not affect CD44-mediated cell adhesion to hyaluronate. Use of site-directed CD44H cDNA mutants with arginine changed to alanine at position 41 indicated that keratan sulfate modification of CD44 modulates hyaluronate adhesion through its B loop domain. These findings suggest that keratan sulfate modification of CD44 may play an important regulatory role in the broad spectrum of biological processes attributed to CD44, including normal development, tumor progression, and lymphocyte function.

Alternative Splicing↗
PubMed1996

Post-translational modification of H-Ras is required for activation of, but not for association with, B-Raf.

B-Raf is regulated by Ras protein and acts as a mitogen-activated protein (MAP) kinase kinase kinase in PC12 cells and brain. Ras protein undergoes a series of post-translational modifications on its C-terminal CAAX motif, and the modifications are critical for its function. To elucidate the role of the post-translational modifications in interaction with, and activation of, B-Raf, we have analyzed a direct association between H-Ras and B-Raf, and constructed an in vitro system for B-Raf activation by H-Ras. By using methods based on inhibition of yeast adenylyl cyclase or RasGAP activity and by in vitro binding assays, we have shown that the segment of B-Raf corresponding to amino acid 1-326 binds directly to H-Ras with a dissociation constant (Kd) comparable to that of Raf-1 and that the binding is not significantly affected by the post-translational modifications. However, when the activity of B-Raf to stimulate MAP kinase was measured by using a cell-free system derived from rat brain cytosol, we observed that the unmodified form of H-Ras possesses an almost negligible activity to activate B-Raf in vitro compared to the fully modified form. H-RasSer-181,184 mutant, which was farnesylated but not palmitoylated, was equally active as the fully modified form. These results indicate that the post-translational modifications, especially farnesylation, are required for H-Ras to activate B-Raf even though they have no apparent effect on the binding properties of H-Ras to B-Raf.

Adenylyl Cyclases↗
PubMed1997

Covalent modification of the regulatory domain irreversibly stimulates cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is regulated by three cytosolic domains, the regulatory domain (R domain) and two nucleotide binding domains. To learn more about how the cytosolic domains regulate channel activity, we used chemical modification to probe their structure. When we applied the sulfhydryl-modifying reagent N-ethylmaleimide (NEM) and other N-substituted maleimides to the cytosolic domains, we found that they rapidly and irreversibly stimulated channel activity. CFTR contains 14 intracellular cysteine residues that might be targets for NEM modification. We identified one, Cys832, that was essential for the response. Cys832 is located in the R domain. Single channel studies showed that NEM stimulated CFTR by increasing the duration of bursts of activity and by shortening the closed interval between bursts. At the single channel level, CFTR in which Cys832 was mutated to alanine behaved identically to wild-type CFTR, except that it failed to respond to NEM. Additional studies showed that NEM modification increased the potency of ATP-mediated stimulation. Previous work has shown that modification of the R domain by phosphorylation, which introduces negative charge, or replacement of multiple serines by negatively charged aspartates stimulates the channel. Our current data show that covalent modification of the R domain with a neutral, hydrophobic adduct at a site that is not phosphorylated can also stimulate CFTR. This finding suggests that an alteration in the conformation of the R domain may be a key feature that regulates channel activity.

Adenosine Triphosphate↗
PubMed1997

The conversion from the dehydrogenase type to the oxidase type of rat liver xanthine dehydrogenase by modification of cysteine residues with fluorodinitrobenzene.

When rat liver xanthine dehydrogenase was incubated with fluorodinitrobenzene (FDNB) at pH 8.5, the total enzyme activity decreased gradually to a limited value of initial activity with modification of two lysine residues in a similar way to the modification of bovine milk xanthine oxidase with FDNB (Nishino, T., Tsushima, K., Hille, R. and Massey, V. (1982) J. Biol. Chem. 257, 7348-7353). After modification with FDNB, the two peptides containing dinitrophenyl-lysine were isolated from the molybdopterin domain after proteolytic digestion and were identified as Lys754 and Lys771 by sequencing the peptides. During the modification of these lysine residues, xanthine dehydrogenase was found to be converted to an oxidase form in the early stage of incubation. Incorporation of the 3H-dinitrophenyl group into enzyme cysteine residues was 0.96 mol per enzyme FAD for 68% conversion to the oxidase form. The modified enzyme was reconverted to the dehydrogenase form by incubation with dithiothreitol with concomitant release of 3H-dinitrophenyl compounds. After modification with 3H-FDNB followed by carboxymethylation under denaturating conditions, the enzyme was digested with proteases. Three 3H-dinitrophenyl-labeled peptides were isolated and sequenced. The modified residues were identified to be Cys535, Cys992 and Cys1324. These residues are conserved among the all known mammalian enzymes, but Cys992 and Cys1324 are not conserved in the chicken enzyme. Cys1324 of the rat enzyme was found not to be involved in the conversion from the dehydrogenase to the oxidase by limited proteolysis experiments, but Cys535 and Cys992 which seemed to be modified alternatively with FDNB appear to be involved in the conversion.

Amino Acid Sequence↗
PubMed1997

Identification of domains on the extrinsic 33-kDa protein possibly involved in electrostatic interaction with photosystem II complex by means of chemical modification.

The extrinsic 33-kDa protein of photosystem II (PSII) was modified with various reagents, and the resulting proteins were checked for the ability to rebind to PSII and to reactivate oxygen evolution. While modification of more than eight carboxyl groups of aspartyl and glutamyl residues with glycine methyl ester did not affect the rebinding and reactivating capabilities, modification of amino groups of lysyl residues with either N-succinimidyl propionate or 2, 4,6-trinitrobenzene sulfonic acid or modification of guanidino groups of arginyl residues with 2,3-butanedione resulted in a loss of rebinding and reactivating capabilities of the 33-kDa protein. Moreover, the number of lysyl and arginyl residues susceptible to modification was significantly decreased when the protein was bound to PSII as compared with when it was free in solution, whereas the number of carboxyl groups modified was little affected. These results suggested that positive charges are important for the electrostatic interaction between the extrinsic 33-kDa protein and PSII intrinsic proteins, whereas negative charges on the protein do not contribute to such interaction. By a combination of protease digestion and mass spectroscopic analysis, the domains of lysyl residues accessible to N-succinimidyl propionate or 2,4, 6-trinitrobenzene sulfonic acid modification only when the 33-kDa protein is free in solution were determined to be Lys4, Lys20, Lys66-Lys76, Lys101, Lys105, Lys130, Lys159, Lys186, and Lys230-Lys236. These domains include those previously reported accessible to N-hydroxysuccinimidobiotin only in solution (Frankel and Bricker (1995) Biochemistry 34, 7492-7497), and may be important for the interaction of the 33-kDa protein with PSII intrinsic proteins.

Amino Acid Sequence↗
PubMed2000

c-Jun and p53 activity is modulated by SUMO-1 modification.

The ubiquitin-related SUMO-1 molecule has been shown recently to modify covalently a number of cellular proteins including IkappaBalpha. SUMO-1 modification was found to antagonize IkappaBalpha ubiquitination and protect it from degradation. Here we identify the transcription factors c-Jun and p53, two well known targets of ubiquitin, as new substrates for SUMO-1 both in vitro and in vivo. In contrast to ubiquitin, SUMO-1 preferentially targets a single lysine residue in c-Jun (Lys-229), and the abrogation of SUMO-1 modification does not compromise its ubiquitination. Activation of Jun NH(2)-terminal kinases, which induces a reduction in c-Jun ubiquitination, similarly decreases SUMO-1 modification. Accordingly, loss of the two major Jun NH(2)-terminal kinase phosphorylation sites in c-Jun, Ser-63 and Ser-73, greatly enhances conjugation by SUMO-1. A SUMO-1- deficient c-JunK229R mutant shows an increased transactivation potential on an AP-1-containing promoter compared with wild-type c-Jun, suggesting that SUMO-1 negatively regulates c-Jun activity. As with c-Jun, SUMO-1 modification of p53 is abrogated by phosphorylation but remains unaltered upon chemical damage to DNA or Mdm2-mediated ubiquitination. The SUMO-1 attachment site in p53 (Lys-386) resides within a region known to regulate the DNA binding activity of the protein. A p53 mutant, defective for SUMO-1 conjugation, shows unaltered ubiquitination but has a slightly impaired apoptotic activity, indicating that modification by SUMO-1 might be important for the full biological activity of p53. Taken together, these data provide a first link between the SUMO-1 conjugation pathway and the regulation of transcription factors.

HeLa Cells↗
PubMed2000

Covalent modification of p73alpha by SUMO-1. Two-hybrid screening with p73 identifies novel SUMO-1-interacting proteins and a SUMO-1 interaction motif.

Two-hybrid screening in yeast with p73alpha isolated SUMO-1 (small ubiquitin-like modifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH. A subset of these proteins contain a common motif, hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the two-hybrid system. We show here that p73alpha, but not p73beta, can be covalently modified by SUMO-1. The major SUMO-1-modified residue in p73alpha is the C-terminal lysine (Lys(627)). The sequence surrounding this lysine conforms to a consensus SUMO-1 modification site b(X)XXhKXE, where b is a basic amino acid. SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1 modification is not required for p73 degradation. SUMO-1 modification does not affect the transcriptional activity of p73alpha on an RGC-luciferase reporter gene in SK-N-AS cells. Instead, SUMO-1 modification may alter the subcellular localization of p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such as those identified here.

Amino Acid Motifs↗