ADP-ribosylation Of Proteins Research Articles

An NAD+ with Dually Modified Adenine for Labeling ADP-Ribosylation-Specific Proteins

Protein adenosine diphosphate (ADP)-ribosylation participates in various pivotal cellular events. Its readers and erasers play key roles in modulating ADP-ribosylation-based signaling pathways. Unambiguous assignments of readers and erasers to individual ADP-ribosylated proteins provide insightful knowledge on ADP-ribosylation biology and require the development of tools and technologies for this goal. Herein, we report the design and the synthesis of a nicotinamide adenine dinucleotide (NAD+) carrying a photoactive and a clickable group. Functioning as a substrate for poly-ADP-ribosylation (PARylation), this NAD+ mimic with dually modified adenine enables covalent crosslinking and labeling of proteins bound to PARylation, representing a new photoaffinity probe for studying this critical post-translational modification.

Abstract 4131674: ADP-Ribosylation In a Mouse Model of Atherosclerosis: a Potential Novel Link Between Dyslipidemia and Inflammation in Cardiovascular Disease

Background: Inflammation and lipid accumulation are major features of atherosclerosis, a leading cause of death and morbidity worldwide. Our previous study recognized ADP-ribosylation, a post-translational modification, as a novel regulator of macrophage activation. We also have established mass spectrometry-based ADP-ribosylation proteomics. Using this technology, we evaluated the completely uncharacterized role of ADP-ribosylation in atherogenesis. We hypothesized that ADP-ribosylated proteins circulate from liver, accumulate in aorta and promote atherogenesis. Methods and Results: We harvested the aorta, liver, and plasma of LDL receptor-deficient ( Ldlr -/- ) mice that were on a regular chow or high-fat diet for 3 or 6 months (n=40/condition). To increase ADP-ribosyl peptide signals in the aorta, we applied our novel recently optimized ion mobility mass spectrometry strategy to generate ADP-ribosylation proteomics data. We analyzed 160 mice aortas and identified 3 APOA1 and 3 APOE ADP-ribosylated peptides in both the aorta and the liver (Fig. A). In addition, these peptides were differentially abundant in the aorta of HFD-fed mice, compared to controls (i.e, APOA1 ARPALEDLR peptide relative abundance (Fig. B)). Using the same mouse plasma, we then validated the presence of ADP-ribosylated APOA1 and ADP-ribosylated APOE in HDL and chylomicron/VLDL/LDL fractions (Western blot), respectively. This finding indicates that classical apolipoproteins circulate as ADP-ribosylated forms, representing a completely novel class of modified apolipoproteins. Immunohistochemistry confirmed the enrichment of aortic lesions in macrophages and ADP-ribosylation signal (5-fold increase, p=0.0006). Conclusions: This work provides the first in vivo evidence that ADP-ribosylation occurs in atherosclerotic lesions, which may originate from the liver via circulating blood (Fig. C). Future studies will examine whether ADP-ribosylation of apolipoproteins, specifically APOA1, alters anti-atherogenic functions of HDL.

Alpha-1 antitrypsin inhibits pertussis toxin

Pertussis (whooping cough) is a vaccine-preventable but re-emerging, highly infectious respiratory disease caused by Bordetella pertussis. There are currently no effective treatments for pertussis, complicating care for non-vaccinated individuals, especially newborns. Disease manifestations are predominantly caused by pertussis toxin (PT), a pivotal virulence factor classified as an ADP-ribosylating AB-type protein toxin. In this work, an unbiased approach using peptide libraries, bioassay-guided fractionation and mass spectrometry revealed α1-antitrypsin (α1AT) as a potent PT inhibitor. Biochemistry-, cell culture- and molecular modeling-based in vitro experimentation demonstrated that the α1AT mode of action is based on blocking PT-binding to the host target cell surface. In the infant mouse model of severe pertussis, α1AT expression was reduced upon infection. Further, systemic administration of α1AT significantly reduced B. pertussis-induced leukocytosis, which is a hallmark of infant infection and major risk factor for fatal pertussis. Taken together our data demonstrates that α1AT is a novel PT inhibitor and that further evaluation and development of α1AT as a therapeutic agent for pertussis is warranted. Importantly, purified α1AT is already in use clinically as an intravenous augmentation therapy for those with genetic α1AT deficiency and could be repurposed to clinical management of pertussis.

A 2-hydroxybutyrate-mediated feedback loop regulates muscular fatigue

Several metabolites have been shown to have independent and at times unexpected biological effects outside of their metabolic pathways. These include succinate, lactate, fumarate, and 2-hydroxyglutarate. 2-Hydroxybutyrate (2HB) is a byproduct of endogenous cysteine synthesis, produced during periods of cellular stress. 2HB rises acutely after exercise; it also rises during infection and is also chronically increased in a number of metabolic disorders. We show here that 2HB inhibits branched-chain aminotransferase enzymes, which in turn triggers a SIRT4-dependent shift in the compartmental abundance of protein ADP-ribosylation. The 2HB-induced decrease in nuclear protein ADP-ribosylation leads to a C/EBPβ-mediated transcriptional response in the branched-chain amino acid degradation pathway. This response to 2HB exposure leads to an improved oxidative capacity in vitro. We found that repeated injection with 2HB can replicate the improvement to oxidative capacity that occurs following exercise training. Together, we show that 2-HB regulates fundamental aspects of skeletal muscle metabolism.

A 2-hydroxybutyrate-mediated feedback loop regulates muscular fatigue.

Several metabolites have been shown to have independent and at times unexpected biological effects outside of their metabolic pathways. These include succinate, lactate, fumarate, and 2-hydroxyglutarate. 2-Hydroxybutyrate (2HB) is a byproduct of endogenous cysteine synthesis, produced during periods of cellular stress. 2HB rises acutely after exercise; it also rises during infection and is also chronically increased in a number of metabolic disorders. We show here that 2HB inhibits branched-chain aminotransferase enzymes, which in turn triggers a SIRT4-dependent shift in the compartmental abundance of protein ADP-ribosylation. The 2HB-induced decrease in nuclear protein ADP-ribosylation leads to a C/EBPβ-mediated transcriptional response in the branched-chain amino acid degradation pathway. This response to 2HB exposure leads to an improved oxidative capacity in vitro. We found that repeated injection with 2HB can replicate the improvement to oxidative capacity that occurs following exercise training. Together, we show that 2-HB regulates fundamental aspects of skeletal muscle metabolism.

An E3 ubiquitin ligase localization screen uncovers DTX2 as a novel ADP-ribosylation-dependent regulator of DNA double-strand break repair

DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: non-homologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response (DDR) relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DDR regulation remains incomplete.Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Amongst these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DTC domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 re-expression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.

Pierisin, Cytotoxic and Apoptosis-Inducing DNA ADP-Ribosylating Protein in Cabbage Butterfly.

Pierisin-1 was serendipitously discovered as a strong cytotoxic and apoptosis-inducing protein from pupae of the cabbage butterfly Pieris rapae against cancer cell lines. This 98-kDa protein consists of the N-terminal region (27 kDa) and C-terminal region (71 kDa), and analysis of their biological function revealed that pierisin-1 binds to cell surface glycosphingolipids on the C-terminal side, is taken up into the cell, and is cleaved to N- and C-terminal portions, where the N-terminal portion mono-ADP-ribosylates the guanine base of DNA in the presence of NAD to induce cellular genetic mutation and apoptosis. Unlike other ADP-ribosyltransferases, pieisin-1 was first found to exhibit DNA mono-ADP-ribosylating activity and show anti-cancer activity in vitro and in vivo against various cancer cell lines. Pierisin-1 was most abundantly produced during the transition from the final larval stage to the pupal stage of the cabbage butterfly, and this production was regulated by ecdysteroid hormones. This suggests that pierisn-1 might play a pivotal role in the process of metamorphosis. Moreover, pierisin-1 could contribute as a defense factor against parasitization and microbial infections in the cabbage butterfly. Pierisin-like proteins in butterflies were shown to be present not only among the subtribe Pierina but also among the subtribes Aporiina and Appiadina, and pierisin-2, -3, and -4 were identified in these butterflies. Furthermore, DNA ADP-ribosylating activities were found in six different edible clams. Understanding of the biological nature of pierisin-1 with DNA mono-ADP-ribosylating activity could open up exciting avenues for research and potential therapeutic applications, making it a subject of great interest in the field of molecular biology and biotechnology.

Histone ADP-ribosylation promotes resistance to PARP inhibitors by facilitating PARP1 release from DNA lesions

Poly(ADP-ribose) polymerase 1 (PARP1) has emerged as a central target for cancer therapies due to the ability of PARP inhibitors to specifically kill tumors deficient for DNA repair by homologous recombination. Upon DNA damage, PARP1 quickly binds to DNA breaks and triggers ADP-ribosylation signaling. ADP-ribosylation is important for the recruitment of various factors to sites of damage, as well as for the timely dissociation of PARP1 from DNA breaks. Indeed, PARP1 becomes trapped at DNA breaks in the presence of PARP inhibitors, a mechanism underlying the cytotoxitiy of these inhibitors. Therefore, any cellular process influencing trapping is thought to impact PARP inhibitor efficiency, potentially leading to acquired resistance in patients treated with these drugs. There are numerous ADP-ribosylation targets after DNA damage, including PARP1 itself as well as histones. While recent findings reported that the automodification of PARP1 promotes its release from the DNA lesions, the potential impact of other ADP-ribosylated proteins on this process remains unknown. Here, we demonstrate that histone ADP-ribosylation is also crucial for the timely dissipation of PARP1 from the lesions, thus contributing to cellular resistance to PARP inhibitors. Considering the crosstalk between ADP-ribosylation and other histone marks, our findings open interesting perspectives for the development of more efficient PARP inhibitor-driven cancer therapies.

PARP14 is regulated by the PARP9/DTX3L complex and promotes interferon γ-induced ADP-ribosylation

Protein ADP-ribosylation plays important but ill-defined roles in antiviral signalling cascades such as the interferon response. Several viruses of clinical interest, including coronaviruses, express hydrolases that reverse ADP-ribosylation catalysed by host enzymes, suggesting an important role for this modification in host-pathogen interactions. However, which ADP-ribosyltransferases mediate host ADP-ribosylation, what proteins and pathways they target and how these modifications affect viral infection and pathogenesis is currently unclear. Here we show that host ADP-ribosyltransferase activity induced by IFNγ signalling depends on PARP14 catalytic activity and that the PARP9/DTX3L complex is required to uphold PARP14 protein levels via post-translational mechanisms. Both the PARP9/DTX3L complex and PARP14 localise to IFNγ-induced cytoplasmic inclusions containing ADP-ribosylated proteins, and both PARP14 itself and DTX3L are likely targets of PARP14 ADP-ribosylation. We provide evidence that these modifications are hydrolysed by the SARS-CoV-2 Nsp3 macrodomain, shedding light on the intricate cross-regulation between IFN-induced ADP-ribosyltransferases and the potential roles of the coronavirus macrodomain in counteracting their activity.

Cholesterol 25-Hydroxylase Protects Against Diabetic Kidney Disease by Regulating ADP Ribosylation Factor 4.

Cholesterol 25-hydroxylase (CH25H), an enzyme involved in cholesterol metabolism, regulates inflammatory responses and lipid metabolism. However, its role in kidney disease is not known. The author found that CH25H transcript is expressed mostly in glomerular and peritubular endothelial cells and that its expression increased in human and mouse diabetic kidneys. Global deletion of Ch25h in Leprdb/db mice aggravated diabetic kidney disease (DKD), which is associated with increased endothelial cell apoptosis. Treatment of 25-hydroxycholesterol (25-HC), the product of CH25H, alleviated kidney injury in Leprdb/db mice. Mechanistically, 25-HC binds to GTP-binding protein ADP-ribosylation factor 4 (ARF4), an essential protein required for maintaining protein transport in the Golgi apparatus. Interestingly, ARF4's GTPase-activating protein ASAP1 is also predominantly expressed in endothelial cells and its expression increased in DKD. Suppression of ARF4 activity by deleting ARF4 or overexpressing ASAP1 results in endothelial cell death. These results indicatethat 25-HC binds ARF4 to inhibit its interaction with ASAP1, and thereby resulting in enhanced ARF4 activity to confer renoprotection. Therefore, treatment of 25-HC improves kidney injury in DKD in part by restoring ARF4 activity to maintain endothelial cell survival. This study provides a novel mechanism and a potential new therapy for DKD.

Abstract 3926: ARF6-dependent endocytic trafficking of the interferon-gamma receptor drives adaptive immune resistance and response to immune checkpoint blockade

Abstract Interferon-gamma (IFNγ)-driven adaptive immune resistance (AIR) in cancer begins at the plasma membrane where cytokine signaling is initiated. Cytotoxic CD8+ T cells secrete IFNγ, eliciting tumor-intrinsic IFNγ signaling and expression of immunosuppressive genes, which are critical for effective therapeutic response to anti-PD-1 and anti-CTLA-4 immune checkpoint blockade (ICB). Although the IFNγ receptor (IFNγR) is expressed in all cell types, its surface density can determine a cell’s responsiveness to ligand. Mechanistic control over this process remains a mystery. Herein we report that the endocytic trafficking protein ADP-Ribosylation Factor 6 (ARF6) is critical for IFNγ-driven AIR during primary tumor progression and in the setting of ICB therapy. The IFNγR1 subunit of the receptor is constitutively internalized in a ligand-independent manner. ARF6 is necessary for endocytic recycling of IFNγR1 and determines the steady-state surface density of the receptor. Loss of ARF6 results in lysosomal degradation of the receptor and controls IFNγR1 protein levels in melanoma, non-small cell lung cancer, colorectal cancer, and triple-negative breast cancer. IFNγ activates ARF6 in parallel with JAK1-STAT1 signaling, suggesting a positive feedback loop that allows tumor cells to dynamically respond to immune attack. Silencing ARF6 reduces tumor intrinsic IFNγ signaling and downstream expression of immunosuppressive genes. In murine melanoma, loss of ARF6 causes resistance to systemic ICB therapy. Likewise, low expression of ARF6 in patient tumors correlates with inferior outcomes with ICB. Our data provide new mechanistic insights into tumor immune escape, defined by ARF6-dependent adaptive immunity, and support that ARF6-dependent endomembrane trafficking of the IFNγ receptor shapes clinical outcomes of ICB therapy. Private Information Citation Format: Yinshen Wee, Junhua Wang, Emily C. Wilson, Coulson P. Rich, Aaron Rogers, Zhongzong Tong, Evelyn DeGroot, Y.N. Vashisht Gopal, Michael Davies, Huseyin Atakan Ekiz, Joshua K.H. Tay, Chris Stubben, Kenneth M. Boucher, Juan M. Oviedo, Keke C. Fairfax, Matthew A. Williams, Sheri L. Holmen, Roger K. Wolff, Allie H. Grossmann. ARF6-dependent endocytic trafficking of the interferon-gamma receptor drives adaptive immune resistance and response to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3926.

Two ferroptosis-specific expressed genes NOX4 and PARP14 are considered as potential biomarkers for the diagnosis and treatment of diabetic retinopathy and atherosclerosis

ObjectivesBoth Diabetic retinopathy (DR) and Atherosclerosis (AS) are common complications in patients with diabetes, and they share major pathophysiological similarities and have a common pathogenesis. Studies performed to date have demonstrated that ferroptosis plays a vital part in the occurrence and development of DR and AS, but its mechanism in the two diseases remains poorly understood.MethodsDR Chip data (GSE60436 and GSE102485) and AS chip data (GSE100927 and GSE57691) were obtained from the Gene Expression Omnibus (GEO) database. The screening of the differential expression genes (DEGs) was analyzed using the limma package, and the genes related to ferroptosis were obtained from the FerrDb V2 database. Two key genes (NOX4 and PARP14) were identified through external datasets validation and receiver operating characteristic (ROC) curve analysis. Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) were used to conduct a functional enrichment analysis, and miRNA-mRNA networks were established. The CIBERSORT algorithm was applied to identify the immune cell infiltration between the disease group and control group. Next, the correlations between key genes and infiltrating immune cells were investigated by the Spearman method. Finally, the correlation between 2 key genes and ferroptosis markers was confirmed.ResultsNine ferroptosis differentially expressed genes (DE-FRGs) between DR and AS were identified in this study. NOX4 and PARP14 were selected as key genes for further analysis by external datasets and ROC curve analysis. The key genes NOX4, PARP14 and their correlated genes (such as CYBA, NOX1, NOX3, CYBB, PARP9, PARP10, and PARP15) are mainly enriched in oxidoreductase activity, protein ADP-ribosylation, superoxide metabolic process, reactive oxygen species metabolic process, PID pathway, and VEGFA-VEGFR2 pathway. A miRNA-mRNA network was constructed, and we got 12 miRNAs correlated with the target gene NOX4, 38 miRNAs correlated with the target gene PARP14. Three common miRNAs (hsa-miR-1-3p, hsa-miR-129-2-3p, and hsa-miR-155-5p) were observed in the network. Immune infiltration analysis displayed that activated B cell, MDSC, and Type 17 T helper cell are the common immune cells involved in the immune infiltration process of DR and AS. The results revealed that there are significant correlations between two key genes and most ferroptosis marker genes no matter in DR or AS.ConclusionFerroptosis-related genes NOX4 and PARP14 may be common biomarkers of DR and AS. Both were associated with immune infiltration in patients with DR and AS. Our data provide a theoretical basis for the early diagnosis and immunotherapy of the two diseases.

A desthiobiotin labelled NAD+ analogue to uncover Poly(ADP-ribose) polymerase 1 protein targets.

ADP-ribosylation is a post-translational modification catalyzed by the enzyme family of polyadenosine diphosphate (ADP)-ribose) polymerases (PARPs). This enzymatic process involves the transfer of single or multiple ADP-ribose molecules onto proteins, utilizing nicotinamide adenine dinucleotide (NAD+ ) as a substrate. It, thus, plays a pivotal role in regulating various biological processes. Unveiling PARP-selective protein targets is crucial for a better understanding of their biological functions. Nonetheless, this task proves challenging due to overlapping targets shared among PARP family members. Therefore, we applied the "bump-and-hole" strategy to modify the nicotinamide binding site of PARP1 by introducing a hydrophobic pocket ("hole"). This PARP1-mutant binds an orthogonal NAD+ (Et-DTB-NAD+ ) containing an ethyl group ("bump") at the nicotinamide moiety. Furthermore, we added a desthiobiotin (DTB) tag directly to the adenosine moiety, enabling affinity enrichment of ADP-ribosylated proteins. Employing this approach, we successfully identified protein targets modified by PARP1 in cell lysate. This strategy expands the arsenal of chemically modified NAD+ analogs available for studying ADP-ribosylation, providing a powerful tool to study these critical post-translational modifications.

Chemical tools for profiling the intracellular ADP-ribosylated proteome

6Yn-Pro is a cell-permeable proprobe successfully undergoing metabolic conversion into 6Yn-NAD+ for subsequent intracellular labelling of ADP-ribosylated proteins.

A Review of PARP-1 Inhibitors: Assessing Emerging Prospects and Tailoring Therapeutic Strategies.

Eukaryotic organisms contain an enzyme family called poly (ADP-ribose) polymerases (PARPs), which is responsible for the poly (ADP-ribosylation) of DNA-binding proteins. PARPs are members of the cell signaling enzyme class. PARP-1, the most common isoform of the PARP family, is responsible for more than 90% of the tasks carried out by the PARP family as a whole. A superfamily consisting of 18 PARPs has been found. In order to synthesize polymers of ADP-ribose (PAR) and nicotinamide, the DNA damage nick monitor PARP-1 requires NAD+ as a substrate. The capability of PARP-1 activation to boost the transcription of proinflammatory genes, its ability to deplete cellular energy pools, which leads to cell malfunction and necrosis, and its involvement as a component in the process of DNA repair are the three consequences of PARP-1 activation that are of particular significance in the process of developing new drugs. As a result, the pharmacological reduction of PARP-1 may result in an increase in the cytotoxicity toward cancer cells.

PARP14 is a writer, reader, and eraser of mono-ADP-ribosylation

PARP14/BAL2 is a large multidomain enzyme involved in signaling pathways with relevance to cancer, inflammation, and infection. Inhibition of its mono-ADP-ribosylating PARP homology domain and its three ADP-ribosyl binding macro domains has been regarded as a potential means of therapeutic intervention. Macrodomains-2 and -3 are known to stably bind to ADP-ribosylated target proteins, but the function of macrodomain-1 has remained somewhat elusive. Here, we used biochemical assays of ADP-ribosylation levels to characterize PARP14 macrodomain-1 and the homologous macrodomain-1 of PARP9. Our results show that both macrodomains display an ADP-ribosyl glycohydrolase activity that is not directed toward specific protein side chains. PARP14 macrodomain-1 is unable to degrade poly(ADP-ribose), the enzymatic product of PARP1. The F926A mutation of PARP14 and the F244A mutation of PARP9 strongly reduced ADP-ribosyl glycohydrolase activity of the respective macrodomains, suggesting mechanistic homology to the Mac1 domain of the SARS-CoV-2 Nsp3 protein. This study adds two new enzymes to the previously known six human ADP-ribosyl glycohydrolases. Our results have key implications for how PARP14 and PARP9 will be studied and how their functions will be understood.

Four of a Kind: A Complete Collection of ADP-Ribosylated Histidine Isosteres Using Cu(I)- and Ru(II)-Catalyzed Click Chemistry.

Adenosine diphosphate ribosylation (ADP-ribosylation) is a crucial post-translational modification involved in important regulatory mechanisms of numerous cellular pathways including histone maintenance and DNA damage repair. To study this modification, well-defined ADP-ribosylated peptides, proteins, and close analogues thereof have been invaluable tools. Recently, proteomics studies have revealed histidine residues to be ADP-ribosylated. We describe here the synthesis of a complete set of triazole-isosteres of ADP-ribosylated histidine to serve as probes for ADP-ribosylating biomachinery. By exploiting Cu(I)- and Ru(II)-catalyzed click chemistry between a propargylglycine building block and an α- or β-configured azidoribose, we have successfully assembled the α- and β-configured 1,4- and 1,5-triazoles, mimicking N(τ)- and N(π)-ADP-ribosylated histidine, respectively. The ribosylated building blocks could be incorporated into a peptide sequence using standard solid-phase peptide synthesis and transformed on resin into the ADP-ribosylated fragments to provide a total of four ADP-ribosyl triazole conjugates, which were evaluated for their chemical and enzymatic stability. The 1,5-triazole analogues mimicking the N(π)-substituted histidines proved susceptible to base-induced epimerization and the ADP-ribosyl α-1,5-triazole linkage could be cleaved by the (ADP-ribosyl)hydrolase ARH3.

The Impact of NAD Bioavailability on DNA Double-Strand Break Repair Capacity in Human Dermal Fibroblasts after Ionizing Radiation.

Nicotinamide adenine dinucleotide (NAD) serves as a substrate for protein deacetylases sirtuins and poly(ADP-ribose) polymerases, which are involved in the regulation of DNA double-strand break (DSB) repair molecular machinery by various mechanisms. However, the impact of NAD bioavailability on DSB repair remains poorly characterized. Herein, using immunocytochemical analysis of γH2AX, a marker for DSB, we investigated the effect of the pharmacological modulation of NAD levels on DSB repair capacity in human dermal fibroblasts exposed to moderate doses of ionizing radiation (IR). We demonstrated that NAD boosting with nicotinamide riboside did not affect the efficiency of DSB elimination after the exposure of cells to IR at 1 Gy. Moreover, even after irradiation at 5 Gy, we did not observe any decrease in intracellular NAD content. We also showed that, when the NAD pool was almost completely depleted by inhibition of its biosynthesis from nicotinamide, cells were still able to eliminate IR-induced DSB, though the activation of ATM kinase, its colocalization with γH2AX and DSB repair capacity were reduced in comparison to cells with normal NAD levels. Our results suggest that NAD-dependent processes, such as protein deacetylation and ADP-ribosylation, are important but not indispensable for DSB repair induced by moderate doses of IR.

Altered cytoplasmic and nuclear ADP-ribosylation levels analyzed with an improved ADP-ribose binder are a prognostic factor in renal cell carcinoma.

ADP-ribosylation (ADPR) of proteins is catalyzed by ADP-ribosyltransferases, which are targeted by inhibitors (i.e. poly(ADP-ribose) polymerase inhibitors [PARPi]). Although renal cell carcinoma (RCC) cells are sensitive in vitro to PARPi, studies on the association between ADPR levels and somatic loss of function mutations in DNA damage repair genes are currently missing. Here we observed, in two clear cell RCC (ccRCC) patient cohorts (n=257 and n=241) stained with an engineered ADP-ribose binding macrodomain (eAf1521), that decreased cytoplasmic ADPR (cyADPR) levels significantly correlated with late tumor stage, high-ISUP (the International Society of Urological Pathology)grade, presence of necrosis, dense lymphocyte infiltration, and worse patient survival (p < 0.01 each). cyADPR proved to be an independent prognostic factor (p=0.001). Comparably, absence of nuclear ADPR staining in ccRCC correlated with absence of PARP1 staining (p < 0.01) and worse patient outcome (p < 0.05). In papillary RCC the absence of cyADPR was also significantly associated with tumor progression and worse patient outcome (p < 0.05 each). To interrogate whether the ADPR status could be associated with genetic alterations in DNA repair, chromatin remodeling, and histone modulation, we performed DNA sequence analysis and identified a significant association of increased ARID1A mutations in ccRCCcyADPR+++/PARP1+ compared with ccRCCcyADPR-/PARP1- (31% versus 4%; p < 0.05). Collectively, our data suggest the prognostic value of nuclear and cytoplasmic ADPR levels in RCC that might be further influenced by genetic alterations.

Protein post-translational modification in SARS-CoV-2 and host interaction.

SARS-CoV-2 can cause lung diseases, such as pneumonia and acute respiratory distress syndrome, and multi-system dysfunction. Post-translational modifications (PTMs) related to SARS-CoV-2 are conservative and pathogenic, and the common PTMs are glycosylation, phosphorylation, and acylation. The glycosylation of SARS-CoV-2 mainly occurs on spike (S) protein, which mediates the entry of the virus into cells through interaction with angiotensin-converting enzyme 2. SARS-CoV-2 utilizes glycans to cover its epitopes and evade the immune response through glycosylation of S protein. Phosphorylation of SARS-CoV-2 nucleocapsid (N) protein improves its selective binding to viral RNA and promotes viral replication and transcription, thereby increasing the load of the virus in the host. Succinylated N and membrane(M) proteins of SARS-CoV-2 synergistically affect virus particle assembly. N protein regulates its affinity for other proteins and the viral genome through acetylation. The acetylated envelope (E) protein of SARS-CoV-2 interacts with bromodomain-containing protein 2/4 to influence the host immune response. Both palmitoylation and myristoylation sites on S protein can affect the virus infectivity. Papain-like protease is a domain of NSP3 that dysregulates host inflammation by deubiquitination and impinges host IFN-I antiviral immune responses by deISGylation. Ubiquitination of ORF7a inhibits host IFN-α signaling by blocking STAT2 phosphorylation. The methylation of N protein can inhibit the formation of host stress granules and promote the binding of N protein to viral RNA, thereby promoting the production of virus particles. NSP3 macrodomain can reverse the ADP-ribosylation of host proteins, and inhibit the cascade immune response with IFN as the core, thereby promoting the intracellular replication of SARS-CoV-2. On the whole, PTMs have fundamental roles in virus entry, replication, particle assembly, and host immune response. Mutations in various SARS-CoV-2 variants, which lead to changes in PTMs at corresponding sites, cause different biological effects. In this paper, we mainly reviewed the effects of PTMs on SARS-CoV-2 and host cells, whose application is to inform the strategies for inhibiting viral infection and facilitating antiviral treatment and vaccine development for COVID-19.