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PubMed2026

Lactylome Reprogramming Mediates Therapeutic Response and Adaptation to Neoadjuvant Chemotherapy in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) exhibits high prevalence in China and poor prognosis despite neoadjuvant chemotherapy (NACT), with significant chemoresistance development. Tumor-associated metabolic reprogramming and NACT-induced cellular stress promote lactate accumulation, which serves as a precursor for lysine lactylation (Kla), a post-translational modification potentially regulating cancer progression. We hypothesized that systematic characterization of the lactylome in response to NACT could reveal critical molecular mechanisms underlying treatment and identify new therapeutic vulnerabilities in ESCC. Herein, through comprehensive proteomic and lactylome profiling of tumor and adjacent normal adjacent tissues from 31 ESCC patients (with or without NACT treatment), we identified 8281 proteins and 1836 Kla sites across 62 samples. NACT induced substantial lactylome alterations with 307 differentially expressed Kla sites predominantly in nonhistone proteins involved in DNA damage response and metabolic pathways. Our data revealed that while NACT-induced suppression of energy metabolism, coupled with upregulated 3-hydroxy-3-methylglutaryl reductase degradation 1 complex expression, may exert potential proapoptotic effects, the activation of ribosome biogenesis and increased nucleoprotein lactylation triggered tumor-protective mechanisms. Mechanistically, we demonstrated that DNA damage and elevated lactate levels induced poly(ADP-ribose) polymerase 1 K654 lactylation, enhancing its enzymatic activity and augmenting poly(ADP-ribosyl)ation of downstream targets, potentially playing a pivotal role in chemotherapy resistance-associated pathways. This comprehensive tissue-level landscape of Kla dynamics in ESCC response to chemotherapy establishes Kla as a critical regulatory mechanism in treatment response, potentially offering novel therapeutic targets and predictive biomarkers for personalized treatment strategies.

Humans↗
PubMed1977

Poly(adenosine diphosphate-ribose) polymerase: the distribution of a chromosome-associated enzyme within the chromatin substructure.

The distribution of a chromatin-bound, nuclear protein modifying enzyme, poly (adenosine diphosphate-ribose) polymerase, and its product, poly(ADP-ribose), among various fractions of sheared and nuclease-digested HeLa cell chromatin has been examined. Epichlorohydrin-tris(hydroxymethyl)aminomethane-cellulose and glycerol gradient fractionation of solubilized chromatin indicated that poly(ADP-ribose)polymerase activity was associated primarily with the template active regions (euchromatin), whereas the transcriptionally inert chromatin fractions were found to contain relatively low levels of ADP-ribosylating activity. When isolated HeLa cell nuclei were digested in situ with micrococcal nuclease and the resultant chromatin was fractionated into nucleosome monomers (v bodies) and oligomers by sucrose gradient centrifugation, only material sedimenting faster than the 11S monomers was found to contain appreciable poly(ADP-ribose) polymerase activity. If, on the other hand, isolated HeLa cell nuclei were first incubated with labeled NAD, the substrate for poly(ADP-ribose) polymerase, prior to the preparation and fractionation of nuclease-digested chromatin, it was found that those chromatin fractions which possess significant poly(ADP-ribose) polymerase activity (nucleosome oligomers) are relatively deficient in the labeled product of this enzyme, and that a considerable portion of the homopolymeric product is ultimately associated with the 11S v bodies. Additional evidence is presented which indicates that the absence of nucleosome monomer-associated poly(ADP-ribose) polymerase activity is not due to the absence of a suitable acceptor on these structures, and that the activity of this enzyme within the chromatin is most probably dependent upon the physical integrity of the oligomeric structures themselves.

Cell Nucleus↗
PubMed2026

PARG inhibition reduces ssDNA levels and limits RPA loading upon replication fork collapse.

Poly(ADP-ribosyl)ation (PARylation) is a transient post-translational modification catalyzed by PARP enzymes and reversed by PARG. PARG inhibition causes sustained PARylation and is being explored as an anticancer strategy, but its cellular consequences remain incompletely understood. Here, we examine how persistent PARylation influences cellular responses to replication stress and DNA damage. We show that sustained PARylation reduces phosphorylated and chromatin-bound RPA most strongly under fork-stalling conditions that progress toward fork collapse. This effect requires PARP1 activity and is restrained by intact ATR-CHK1 signaling, as checkpoint inhibition renders otherwise resistant cells permissive for PARG inhibitor-associated phosphorylated RPA loss from the chromatin. The reduction of RPA phosphorylation is not dependent on BRCA1 and it is not accompanied by increased RAD51 loading. Instead, reduced chromatin-bound RPA coincides with decreased exposed ssDNA. Our results identify a checkpoint-dependent fork-collapse state in which sustained PARylation limits ssDNA and RPA levels.

Replication Protein A↗
PubMed1976

Effect of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 on translocation.

1. The effect of elongation factor 2 (EF 2) and of adenosine diphosphate-ribosylated elongation factor 2 (ADP-ribosyl-EF 2) on the shift of endogenous peptidyl-tRNA from the A to the P site of rat liver ribosomes (measured by the peptidyl-puromycin reaction) and on the release of deacylated tRNA (measured by aminoacylation) was investigated. 2. Limiting amounts of EF2, pre-bound or added to ribosomes, catalyse the shift of peptidyl-tRNA in the presence of GPT; when the enzyme is added in substrate amounts GMP-P(CH2)P [guanosine (beta, gamma-methylene)triphosphate] can partially replace GTP. ADP-ribosyl-EF 2 has no effect on the shift of peptidyl-tRNA when present in catalytic amounts, but becomes almost as effective as EF 2 when added in substrate amounts together with GTP; GMP-P(CH2)P cannot replace GTP. 3. The release of deacylated tRNA is induced only by substrate amounts of added EF 2 and also occurs in the absence of guanine nucleotides. In this reaction ADP-ribosyl-EF 2 is only 25% as effective as EF 2 in the absence of added nucleotide, but becomes 60-80% as effective in the presence of GTP or GMP-P(CH2)P. 4. The results obtained on protein-synthesizing systems are consistent with the hypothesis that ADP-ribosyl-EF 2 can operate a single round of translocation followed by binding of aminoacyl-tRNA and peptide-bond formation. 5. From the data obtained with the native enzyme it is concluded that the two moments of translocation require different conditions of interaction of EF 2 with ribosomes; it is suggested that the shift of peptidyl-tRNA is catalysed by EF 2 pre-bound to ribosomes, and that the release of tRNA is induced by a second molecule of interacting EF 2. The hydrolysis of GTP would be required for the release of pre-bound EF 2 from ribosomes. 5. The inhibition of the utilization of limiting amounts of EF 2 on ADP-ribosylation is very likely the consequence of a concomitant decrease in the rate of association and dissociation of the enzyme from ribosomes.

Adenosine Diphosphate Sugars↗
PubMed1977

Adenosine diphosphate ribosylated histones.

When rat liver nuclei were incubated with [adenine-3H]NAD, besides histone 1, histone 2A and especially histone 2B accepted 3H radioactivity. 3H radioactivity was also found on the non-histone proteins and on the small amounts of histones 1 and 3 released into the supernatant during incubation. [14C]Adenine uptake in vivo by liver and thymus nuclei showed radioactivity in histones 1 and 3. After digestion with Pronase and leucine aminopeptidase 14C- or 32P-labelled histone 3 released a serine phosphate-containing nucleotide, which on acid hydrolysis yielded ADP-ribose and serine phosphate. Serine phosphate was also found in the material from the nucleotide peaks from histones 2A and 2B. ADP-ribosylated histones 1 and 3 were more easily released from nuclei than their unmodified forms and showed higher [32P]Pi and [3H]lysine uptakes in vivo [Ord & Stocken (1975) FEBS Meet. Proc. 34, 113-125].

Adenine↗
PubMed1977

Phosphorylation of rat thymus histones, its control and the effects thereon of gamma-irradiation.

The phosphate content of rat thymus histones was determined. As expected for a replicating tissue, histones 1 and 2B were more phosphorylated and had higher 32P uptakes than did histones from resting liver nuclei; the other histones all showed 32P uptake, but the phosphate content and uptake of histone 2A was about half that for liver histone 2A. When thymus nuclei were incubated in a slightly hypo-osmotic medium, non-histone proteins and phosphorylated histones were released into solution; this was enhanced if ATP was present in the medium. [gamma-32P]ATP was incorporated into non-histone proteins, including protein P1, and into the ADP-ribosylated form of histone 1; negligible 32P was incprporated into the other, bound, histones. Histones 1 and 2B added to the incubation medium were extensively, and histones 2A and 4 slightly, phosphorylated. Histones released by increasing the ionic strength of the medium were phosphorylated. Added lysozyme and cytochrome c were neither bound nor phosphorylated, but added non-histone protein P1 was phosphorylated, causing other histones to be released from the nuclei, especially histones 2A and 3. The released histones were phosphorylated. gamma-Irradiation decreased 32P uptake into the non-ADP-ribosylated histones 1 and 4; phosphorylation of histone 1 in vitro was unaffected. The importance of non-histone proteins, ATP availability and nuclear protein kinases to the control of histone phosphorylation in vivo is discussed.

Adenosine Triphosphate↗
PubMed1976

A ribosome-dependent GTPase from yeast distinct from elongation factor 2.

Three proteins required for poly(U)-directed polyphenylalanine synthesis have been separated from yeast. Two of the factors correspond to the elongation factors 1 and 2 described for other eukaryotic systems, according to the criteria of phenylalanyl-tRNA binding and diphtheria toxin-catalyzed ADP-ribosylation. The third protein, while absolutely required for polyphenylalanine synthesis, was a more active ribosome-dependent GTPase than elongation factor 2.

Adenosine Diphosphate↗
PubMed1976

Hydrolysis of nicotinamide adenine dinucleotide by choleragen and its A protomer: possible role in the activation of adenylate cyclase.

Choleragen and the isolated A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. The protein with NADase activity (NAD nucleosidase; NAD glycohydrolase, EC 3-2-2-5) migrated on polyacrylamide gels with choleragen, and chromatographed on Bio-Gel P-60 columns with the A protomer. The NADase activity of choleragen and of the A protomer was increased markedly in acetate and phosphate buffers, and enhanced over 10-fold by dithiothreitol in high concentration. NAD hydrolysis was proportional to choleragen concentration; the Michaelis constant for NAD was about 4 mM with both choleragen and the A protomer. The demonstration that the A protomer of choleragen catalyzes an enzymatic reaction involving activation of the ribosyl-nicotinamide bond of NAD, a reaction analogols to those catalyzed by diphtheria toxin, supports the hypothesis that activation of adenylate cyclase by choleragen involves the ADP-ribosylation of an appropriate acceptor protein.

Adenylyl Cyclases↗
PubMed1977

Transfer of ADP-ribose from NAD to choleragen: a subunit acts as catalyst and acceptor protein.

Choleragen selectively incorporates 3H from [3H]NAD labeled on the adenosine moiety and not 14C from [14C]NAD labeled on the nicotinamide moiety. This reaction does not require protein in addition to choleragen. Incorporation of isotope does not proceed at 4 degrees, requires dithiothreitol, is stable after extensive washing with cold trichloroacetic acid, and is decreased 80% by boiling in trichloroacetic acid. Studies with the A and B subunits of choleragen show that the A subunit catalyzes ADP-ribosylation and serves as an acceptor protein. The B subunit does not show catalytic or acceptor activity. We conclude that choleragen and its A subunit catalyze the hydrolysis of NAD and the enzymatic transfer of ADP-ribose to the A subunit.

Adenosine Diphosphate Sugars↗
PubMed1978

Mechanism of cholera toxin action: covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system.

Treatment of pigeon erythrocyte membranes with cholera toxin and NAD(+) enhanced the GTP stimulation and suppressed the F(-) activation of the adenylate cylase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. In the presence of NAD(+) labeled with (32)P in the AMP moiety the toxin catalyzed the covalent incorporation of radioactivity into membrane proteins with molecular weights (M(r)s) of 200,000, 86,000, and 42,000. Extraction of toxin-treated membranes with Lubrol PX followed by affinity chromatography on a GTP-Sepharose column resulted in a 200-fold purification of the 42,000-M(r) labeled protein and in its complete separation from the other labeled proteins. The fraction containing the purified GTP-binding component from toxin-treated membranes conferred an enhanced GTP-stimulated activity on adenylate cyclase solubilized from nontreated membranes. Likewise, the addition of GTP-binding fraction from nontreated membranes to an enzyme solubilized from toxin-treated membranes restored F(-) stimulation of the adenylate cyclase. The toxin-induced modification of adenylate cyclase and the incorporation of radioactivity into the 42,000-M(r) protein were partially reversed upon incubation with toxin and nicotinamide at pH 6.1. The results indicate that cholera toxin affects the adenylate cyclase system by catalyzing an ADP-ribosylation of the 42,000-M(r) component bearing the guanyl nucleotide regulatory site.

Adenosine Diphosphate Sugars↗
PubMed1978

A nuclear protein-modifying enzyme is responsive to ordered chromatin structure.

Poly (ADP-ribose) polymerase, a nuclear protein-modifying enzyme, binds to the internucleosomal linker region of chromatin, although it modifies certain core nucleosomal histones in addition to histone H1. The activity per unit of DNA chromatin changes with the nucleosome repeat number. It reaches a maximum on chromatin of 8-10 nucleosomes in length. As the complexity of chromatin with respect to nucleosome repeat number and compactness increases, a decline and stabilization of specific activity is noted. The difference in specific activity is maintained through resedimentation and dialysis of particles. It does not appear due to differences in polymer chain length or differential degradation of poly (ADP-ribose). The data suggest a relationship between ADP-ribosylation and chromatin organization and vice versa.

Binding Sites↗
PubMed1977

Incidence of exotoxin production by Pseudomonas species.

Pseudomonas aeruginosa exotoxin A has been shown to catalyze the transfer of the adenosine 5'-diphosphate (ADP)-ribose moiety of nicotinamide adenine dinucleotide onto elongation factor 2, resulting in the inhibition of mammalian protein synthesis. The enzymatic activity (ADP-ribosyl [ADPR]-transferase) is thought to account for the toxicity of exotoxin A. The distribution of the expression of exotoxin A within Pseudomonas species was examined. Laboratory strains as well as clinical isolates of Pseudomonas aeruginosa were tested. The production of exotoxin A was determined by assaying for ADPR-transferase activity in dialyzed frozen (-20 degrees C) and thawed cell-free supernatants from 22-h cultures or in 10-fold-concentrated supernatants. In addition, toxin production was detected immunologically using a modified Elek test. Exotoxin A production was detected in approximately 90% of the 111 isolates of P. aeruginosa. In contrast, none of the other species of Pseudomonas examined produced exotoxin A detectable by either ADPR-transferase activity or immunological reactivity.

Epitopes↗
PubMed1977

Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity.

The sensitivities of 21 mammalian cell lines to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae were measured. Each line exhibited 1-4 log differences in sensitivities to the two toxins. No species-specific sensitivities were noted for Pseudomonas exotoxin while diphtheria exotoxin was most potent in cells of monkey origin, followed by human and hamster cells. Rat- and mouse-derived cell lines were very insensitive to diphtheria exotoxin. The rates of cellular intoxication by both toxins exhibited apparent first-order kinetics and were indistinguishable from one another when equipotent doses were used. Our preparation of diphtheria exotoxin appeared to have a slightly higher ADP-ribosylating efficiency than did Pseudomonas toxin. However, neither toxin exhibited cell line-specific differences in ribosylating efficiencies which could have explained the wide range in potencies for intact cells. Our results suggest that there are significant differences in the mechanisms of cellular intoxication by Pseudomonas and diphtheria exotoxins and that these differences probably exist in the attachment or internalization stages of toxin action.

Animals↗
PubMed2026

ARL6IP1 Inhibits Breast Cancer Tumor Progression by Targeting OLFM4 to Regulate Glycolysis.

INTRODUCTION: ARL6IP1 has been linked to cancer progression, but its precise role in BC, particularly in metabolism and its interaction with an OLFM4, remains unclear. AIMS: This study aimed to investigate the role of ADP-ribosylation factor-like 6 interacting protein 1 (ARL6IP1) in breast cancer (BC) cell behavior and metabolism and explore its interaction with an olfactomedin-4 (OLFM4) as a potential therapeutic target. OBJECTIVE: The objective of this study was to determine the effects of ARL6IP1 knockdown on BC cell proliferation, invasion, migration, apoptosis, oxidative stress, and glycolysis. Additionally, this study also explored the interaction between ARL6IP1 and OLFM4 and their combined role in BC progression and metabolism. METHODS: Key gene modules in the GSE73540 dataset were identified through weighted gene co-expression network analysis (WGCNA). Three BC-related datasets (GSE73540, GSE22820, and GSE36295) and The Cancer Genome Atlas (TCGA) were applied for additional examination of differentially expressed genes (DEGs). Intersection analysis selected ARL6IP1 as a hub gene for prognostic analysis. In vitro experiments investigated how ARL6IP1 knockdown influences BC cell proliferation, invasion, migration, apoptosis, epithelial-mesenchymal transition (EMT), oxidative stress, and glycolysis. The connection between ARL6IP1 and an OLFM4 was confirmed using Co-immunoprecipitation (Co-IP), and their roles in BC tumor progression and glycolysis were evaluated. RESULTS: ARL6IP1 was elevated in BC datasets and linked with poor BC prognosis. Experiments demonstrated that knockdown of ARL6IP1 significantly reduced BC cell growth while promoting apoptosis and oxidative stress. Besides, ARL6IP1 knockdown reduced glycolysis, as manifested by decreased extracellular acidification rate (ECAR), glucose consumption, adenosine triphosphate (ATP) levels, and lactate production while increasing mitochondrial respiration (OCR). Co-IP validated the connection between ARL6IP1 and OLFM4, and OLFM4 overexpression partially counteracted the suppression of glycolysis and cell behavior resulting from ARL6IP1 knockdown. CONCLUSION: ARL6IP1 is a critical regulator of BC progression, influencing glycolysis, mitochondrial function, and key cellular behaviors. Targeting the ARL6IP1-OLFM4 axis offers a promising therapeutic strategy for managing BC.

Humans↗
PubMed1977

Interaction of elongation factor 2 from wheat germ with guanosine nucleotides and ribosomes.

1. The amino acid composition of wheat germ EF2 differs to some extent from that of elongation factors from mammals and bacteria. 2. The purified wheat germ EF2, similarly as the factors from other sources, is active in the: EF1-dependent polymerization of phenylalanine; ribosome-dependent GTP hydrolysis; binding of guanosine nucleotides; and ADP-ribosylation in the presence of diphtheria toxin. Fusidic acid at a concentration of 1 mM inhibits all these EF2-dependent reactions. 3. Diphtheria toxin in the presence of NAD+ inhibits polymerization of phenylalanine but does not effect GTP binding to EF2. 4. Binding of GDP to wheat germ EF2 is inhibited by ribosomes. During interaction with ribosomes, GTP in EF2-GTP complex is rapidly hydrolysed to GDP. Both GTP and 5'-guanylmethylenediphosphonate competitively inhibit formation of the ribosome-EF2-GDP complex due to the replacement of GDP from the complex. The latter is stabilized by fusidic acid.

Adenosine Diphosphate↗
PubMed1977

Purification and properties of poly(adenosine diphosphate ribose) synthetase.

Poly(ADP-ribose) synthetase has been purified approximately 5000-fold from rat liver nuclei. The activity of the purified enzyme is absolutely dependent upon the presence of native or synthetic DNA, and the further addition of histone(s) stimulates the activity 3- to 5-fold. When the ADP-ribosylated material synthesized in the absence or presence of various histones is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the major product in all cases migrates between histones H1 and H3-H2B with the same RF value of 0.58 relative to the marker dye. No ADP-ribose was found to co-electrophorese with any of thehistones. The addition of histones does not affect the chain number of the poly(ADP-ribose) synthesized but does result in an increase in the average chain length of the polymer. In the presence of histones, the Km for NAD+ decreases from 80 micron to 25 micron and the Vmax doubles. These results indicate that, in the purified poly(ADP-ribose) synthetase system, histones are not ADP-robosylated but act as allosteric activators.

Animals↗
PubMed1979

ADP ribosylation of rat liver nucleosomal core histones.

When nucleosomal core histones were isolated from rat liver nuclei incubated with [14C]NAD+ and fractionated into the individual components (H2A, H2B, H3, and H4), [14C]adenosine diphosphate ribose (ADP-Rib) was found to be associated with all of them. However, while about 15% of the H2B molecules were modified, less than 2% of the other fractions contained radioactive ADP-Rib. The nucleotide attached to H2B was identified as a single monomer of ADP-Rib. On subjectint H2B to electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid, one single band of H2B with 5% less mobility than the unomdified control was obtained. The linkage between H2B and ADP-Rib was rapidly hydrolyzed with 0.1 N NaOH or with 1 M neutral hydroxylamine. Hydrolysis of ADP-ribosylated H2B with trypsin generated a single peptide linked to ADP-Rib, which corresponded to the sequence Pro-Glu-Pro-Ala-Lys. We were able to dansylate the NH2-terminal proline, which proved that the imino group of this amino acid was not substituted. These findings, together with the chemical properties of the linkage, which were typical of those of an ester-like bond, strongly suggest that the ADP-Rib residue was linked to the gamma-COOH group of the glutamic acid in position 2 of H2B.

Adenosine Diphosphate Sugars↗
PubMed2026

Genetically predicted associations between blood cell perturbation responses and bronchiectasis through immune mediation: A Mendelian Randomization study.

BACKGROUND: Bronchiectasis is a chronic airway disease characterized by persistent inflammation and structural damage, with substantial clinical and etiologic heterogeneity. Although previous studies have identified associations between blood cells and bronchiectasis, the causal relationships remain unclear. Moreover, the mechanisms underlying blood cell perturbation responses and their potential mediation by immune cells in disease progression are largely unexplored. METHODS: Two-sample Mendelian randomization (MR) analysis was used to explore genetically predicted associations among immune cell traits, blood cell perturbation response phenotypes, and bronchiectasis, based on genome-wide association study summary data. Mediation MR analysis was further applied to assess whether immune cells mediate these associations. Multiple sensitivity analyses, including tests for heterogeneity and horizontal pleiotropy, were performed to evaluate the validity and robustness. RESULTS: Five blood cell perturbation response phenotypes and twenty-nine immune cell traits showed significant genetically predicted associations with bronchiectasis. Mediation analysis showed that natural killer (NK) cell absolute count partially mediated the causal effect between the eosinophil perturbation response and bronchiectasis, with a mediation proportion of 9.626%. CD38 on transitional B cells mediated the causal effect between the monocyte perturbation response and bronchiectasis, with a mediation proportion of 10.580%. Additionally, CD45 on NK cells played a mediating role in the association between the white blood cell perturbation response and bronchiectasis, with a mediation proportion of 10.651%. CONCLUSION: This study systematically explores genetically predicted associations between blood cell perturbation responses and bronchiectasis and highlights potential immune-mediated pathways. These exploratory findings provide novel genetic insights into the pathogenesis of bronchiectasis and identify potential therapeutic targets for future strategies.

Humans↗