Posttranslational Modification Enzymes Research Articles

PAD4 and Its Inhibitors in Cancer Progression and Prognosis.

The systemic spread of malignancies and the risk of cancer-associated thrombosis are major clinical challenges in cancer therapy worldwide. As an important post-translational modification enzyme, peptidyl arginine deiminase 4 (PAD4) could mediate the citrullination of protein in different components (including nucleus and cytoplasm, etc.) of a variety of cells (tumor cells, neutrophils, macrophages, etc.), thus participating in gene regulation, neutrophil extracellular trap (NET) and macrophage extracellular trap (MET). Thereby, PAD4 plays an important role in enhancing the growth of primary tumors and facilitating the distant metastasis of cancer cells. In addition, it is related to the formation of cancer-associated thrombosis. Therefore, the development of PAD4-specific inhibitors may be a promising strategy for treating cancer, and it may improve patient prognosis. In this review, we describe PAD4 involvement in gene regulation, protein citrullination, and NET formation. We also discuss its potential role in cancer and cancer-associated thrombosis, and we summarize the development and application of PAD4 inhibitors.

SN-38 Sensitizes BRCA-Proficient Ovarian Cancers to PARP Inhibitors through Inhibiting Homologous Recombination Repair.

As a multifunctional protein posttranslational modification enzyme in eukaryotic cells, Poly-ADP-ribose polymerase (PARP) acts as a DNA damage sensor, which helps to repair DNA damage through recruiting repair proteins to the DNA break sites. PARP inhibitors offer a significant clinical benefit for ovarian cancer with BRCA1/2 mutations. However, the majority of ovarian cancer patients harbor wild-type (WT) BRCA1/2 status, which narrows its clinical application. Here, we identified a small compound, SN-38, a CPT analog, which sensitizes BRCA-proficient ovarian cancer cells to PARP inhibitor treatment by inhibiting homologous recombination (HR) repair. SN-38 treatment greatly enhanced PARP inhibitor olaparib induced DNA double-strand breaks (DSBs) and DNA replication stress. Meanwhile, the combination of SN-38 and olaparib synergistically induced apoptosis in ovarian cancer. Furthermore, combination administration of SN-38 and olaparib induced synergistic antitumor efficacy in an ovarian cancer xenograft model in vivo. Therefore, our study provides a novel therapeutic strategy to optimize PARP inhibitor therapy for patients with BRCA-proficient ovarian cancer.

Study on the regulation of immunity by palmitoylation

Palmitoylation is a reversible enzymatic post-translational modification of a protein, where the palmitoyl chain connects to cysteine residues via thioester bonds. Palmitoylation affects protein function by affecting protein activity, stability, protein transport, and protein-protein interactions. Palmitoylation occurs in lymphocytes that are critical to both innate and acquired immune responses. Palmitoylation can also occur in the receptors of immune cells, thereby regulating immunity. In this paper, the role of palmitoylation in immunity is summarized by better combing the immune-related signaling pathways affected by palmitoylation.

Accurate Models of Substrate Preferences of Post-Translational Modification Enzymes from a Combination of mRNA Display and Deep Learning.

Promiscuous post-translational modification (PTM) enzymes often display nonobvious substrate preferences by acting on diverse yet well-defined sets of peptides and/or proteins. Understanding of substrate fitness landscapes for PTM enzymes is important in many areas of contemporary science, including natural product biosynthesis, molecular biology, and biotechnology. Here, we report an integrated platform for accurate profiling of substrate preferences for PTM enzymes. The platform features (i) a combination of mRNA display with next-generation sequencing as an ultrahigh throughput technique for data acquisition and (ii) deep learning for data analysis. The high accuracy (>0.99 in each of two studies) of the resulting deep learning models enables comprehensive analysis of enzymatic substrate preferences. The models can quantify fitness across sequence space, map modification sites, and identify important amino acids in the substrate. To benchmark the platform, we performed profiling of a Ser dehydratase (LazBF) and a Cys/Ser cyclodehydratase (LazDEF), two enzymes from the lactazole biosynthesis pathway. In both studies, our results point to complex enzymatic preferences, which, particularly for LazBF, cannot be reduced to a set of simple rules. The ability of the constructed models to dissect such complexity suggests that the developed platform can facilitate a wider study of PTM enzymes.

Control of Nucleophile Chemoselectivity in Cyanobactin YcaO Heterocyclases PatD and TruD.

Members of the YcaO superfamily are among the most common post-translational modification enzymes in natural product biosynthesis, with wide usage in biotechnology and synthetic biology applications. Here, we use domain-swapped chimeras and discovered unstructured regions in cyanobactin YcaOs that guide interactions with the substrates, governing access to interior amino acids in the substrates and explaining the chemoselectivity between PatD and TruD. These results define how the cyanobactin heterocyclases modify exceptionally sequence diverse substrates, yet with a high degree of positional and nucleophile selectivity.

Specific and sensitive GC–MS analysis of hypusine, Nε-(4-amino-2-hydroxybutyl)lysine, a biomarker of hypusinated eukaryotic initiation factor eIF5A, and its application to the bi-ethnic ASOS study

Hypusination is a unique two-step enzymatic post-translational modification of the Nε-amino group of lysine-50 of the eukaryotic initiation factor 5A (eIF5A). We developed a specific and sensitive gas chromatography–mass spectrometry (GC–MS) method for the measurement of biological hypusine (Hyp), i.e., Nε-(4-amino-2-hydroxybutyl)lysine. The method includes a two-step derivatization of Hyp: first esterification with 2 M HCl in CH3OH (60 min, 80 °C) to the methyl ester (Me) and then acylation with penta-fluoro-propionic (PFP) anhydride in ethyl acetate (30 min, 65 °C). Esterification with 2 M HCl in CD3OD was used to prepare the internal standard. The major derivatization product was identified as the un-labelled (d0Me) and the deuterium-labelled methyl esters (d3Me) derivatives: d0Me-Hyp-(PFP)5 and d3Me-Hyp-(PFP)5, respectively. Negative-ion chemical ionization generated the most intense ions with m/z 811 for d0Me-Hyp-(PFP)5 and m/z 814 for the internal standard d3Me-Hyp-(PFP)5. Selected-ion monitoring of m/z 811 and m/z 814 was used in quantitative analyses. Free Hyp was found in spot urine samples (10 µL) of two healthy subjects at 0.60 µM (0.29 µmol Hyp/mmol creatinine) in the female and 1.80 µM (0.19 µmol Hyp/mmol creatinine) in the male subject. The mean accuracy of the method in these urine samples spiked with 1–5 µM Hyp was 91–94%. The limit of detection (LOD) of the method is 1.4 fmol Hyp. The method was applied to measure the urinary excretion rates of Hyp in healthy black (n = 38, age 7.8 ± 0.7 years) and white (n = 41, age 7.7 ± 1.0 years) boys of the Arterial Stiffness in Offspring Study (ASOS). The Hyp concentrations were 3.55 [2.68–5.31] µM (range 0.54–9.84 µM) in the black boys and 3.87 [2.95–5.06] µM (range 1.0–11.7 µM) in the white boys (P = 0.64). The creatinine-corrected excretion rates were 0.25 [0.20–0.29] µmol/mmol (range 0.11–0.36 µmol/mmol) in the black boys and 0.26 [0.21–0.30] µmol/mmol (range 0.10–0.45 µmol/mmol) in the white boys (P = 0.82). These results suggest that there is no ethnic-related difference in the ASOS population in the eIF5A modification. Remarkable differences were found between black and white boys with respect to correlations of urinary Hyp with amino acids and advanced glycation end-products of Lys, Arg and Cys. Deoxyhypusine, formally the direct precursor of Hyp, seems not to be excreted in the urine by healthy subjects.

ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NF\u03baB activation

Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.

Single nanoparticle tracking in the endoplasmic reticulum lumen

Molecular crowding in the endoplasmic reticulum (ER) lumen dictates the rates at which folding chaperones, post-translational modification enzymes, and export factors diffuse and associate with nascent secretory proteins. Changes in ER morphology and ER stress can alter crowding in the ER, impacting secretory proteins’ ability to encounter enzymes and factors necessary for secretion. Existing measurements of luminal diffusion have relied on bulk flow measurements (using fluorescence recovery after photobleaching) or single particle tracking of membrane-bound proteins.

Fluorescence Lifetime Imaging Probes for Cell-Based Measurements of Enzyme Activity.

Posttranslational modification (PTM) enzymes are important modulators of protein structure and function. They typically act by chemically modifying amino acids, often on side chain functional groups, to change the physiochemical landscape of the protein and thus its biophysical behavior. In particular, protein kinases are enzymes that transfer phosphate from ATP to serine, threonine, or tyrosine in protein substrates. They are key regulators of vital cellular pathways such as survival, proliferation, and apoptosis, and their dysregulation in the context of cancer has been widely investigated for the purpose of development of anticancer drugs. However, several critical questions pertaining to their physiology, such as heterogeneity of kinase signaling within and between cells, and other factors that may play into the mechanisms of drug resistance, remain unanswered. Many of the current strategies to measure kinase activity lack the scope, subcellular resolution, and real-time monitoring ability needed to obtain the type of information needed about their dynamics and localization in cells. While FRET-based biosensors are capable of dynamic single cell imaging, their applications can be limited by difficulties in multiplexing and the inherent inadequacies of steady state measurements. In this chapter, we describe our fluorescence lifetime imaging microscopy (FLIM) probe technology in which peptide kinase substrates, linked to cell-penetrating peptides and labeled with small molecule fluorophores, are used to report kinase activity through time-resolved fluorescence imaging to visualize and quantify changes to the probe's fluorescence lifetime. These can be multiplexed for more than one kinase at a time, and interpretation is not affected by differences in local intensity due to probe uptake and distribution or photobleaching. With careful choice of peptide substrate(s), fluorophore label, and imaging set-up, high specificity and spatiotemporal resolution can be achieved. Due to the mechanism by which the lifetime change occurs, this approach is compatible with other PTMs (such as acetylation, methylation), and so the considerations for kinase FLIM probe design described in this chapter should be broadly applicable for other PTMs as well.

Abstract 11607: Protein Citrullination Landscape of Human Coronary Atherosclerosis

Introduction: Recently, an irreversible enzymatic post-translational modification, citrullination, has been shown to be present in the cardiovascular system (e.g. aorta). Citrullination, in which arginine is converted to citrulline, can alter protein structure and function, and produce autoantigens Hypothesis: Emerging evidence suggest a potential role for autoimmunity in the pathogenesis of atherosclerosis. However, it remains unknown whether of post-translational citrullination of arterial proteins contributes to local inflammatory processes that initiate or accelerate the atherosclerotic process. The aim of this study was to investigate changes in protein citrullination in human left anterior descending (LAD) coronary artery specimens with and without early atherosclerosis obtained from autopsied young adults with no clinical manifestations of coronary disease. Methods: Segments of the mid-LAD from 74 autopsied individuals (age range:15-55 yrs., 75% male, 67% White) were graded by a pathologist for percent of surface area involved with fatty streaks (FS) or fibrous plaque (FP). The frozen samples were homogenized and digested with Lys-C for subsequent proteomic mass-spectrometry using a data-independent acquisition (DIA) proteomic strategy with a hyper-citrullinated spectral library approach and our published CitFinder software. Results: We found 133 differentially expressed proteins (FDR<0.05) between control, FS and FP groups. Among them, 19 citrullinated proteins with 26 modified amino acid residues were identified. Citrullinated peptides from fibrinogen alpha chain (R84) and transgelin (R146) were upregulated in FP samples in comparison to FS and normal samples. Similarly, the citrullination site (R269) on alpha-enolase was significantly increased in the FP samples compared to normal. Interestingly, citrullination (R110) on myosin light polypeptide 6 was downregulated in FP samples. Conclusions: These data represent the most comprehensive interrogation of citrullinated proteins in human arterial samples to-date and suggest a possible association between this modification of several key proteins involved in tight junction, vascular smooth muscle contraction and inflammation and early atherosclerosis.

Heterogeneous sensing of post-translational modification enzymes by integrating the advantage of homogeneous analysis

Heterogeneous analysis has great application prospects in the detection of post-translational modification (PTM) enzymes with the advantages of signal enhancement, less sample demand, and high sensitivity and selectivity. Nevertheless, once the substrate was fixed on a solid interface, the steric hindrance might limit the approaching of catalytic center to the substrate, thus reducing the efficiency of PTM. Herein, we suggested that the avidin-modified interface could be used to develop heterogeneous sensing platforms with biotin-labeled substrates as the probes, in which the enzymatic PTM was performed in solution and the heterogeneous assay was conducted on a solid surface. The sensing strategy integrates the advantages but overcomes the defects of both homogeneous and heterogeneous assays. Protein kinase A (PKA) and histone acetyltransferase (HAT) were determined as the examples by using sequence-specific peptide substrates. The signal changes were monitored by HRP-based colorimetric assay and antibody-amplified surface plasmon resonance (SPR). The methods were used for analysis of cell lysates and evaluation of inhibition efficiency with satisfactory results. The strategy can be used for the detection of a variety of biological enzymes and provide a new idea for the design of various heterogeneous biosensors. Thus, this work should be of great significance to the popularization and practical application of biosensors.

Profiling sirtuin activity using Copper-free click chemistry

The mammalian sirtuins are a group of posttranslational modification enzymes that remove acyl modifications from lysine residues in an NAD+-dependent manner. Although initially proposed as histone deacetylases (HDACs), they are now known to target other cellular enzymes and proteins as well. Sirtuin-catalyzed simple amide hydrolysis has profound biological consequences including suppression of gene expression, promotion of DNA damage repair, and regulation of glucose and lipid metabolism. Human sirtuins have been intensively pursued by both academia and industry as potential therapeutic targets for the treatment of diseases such as cancer and neurodegeneration. To gain a better understanding of their roles in various cellular events, innovative chemical probes are highly sought after. This current study focuses on the development of activity-based chemical probes (ABPs) for the profiling of sirtuin activity in biological samples. Cyclooctyne-containing and azido-containing probes were synthesized to enable the subsequent copper-free “click” conjugation to either a fluorophore or biotin. The two groups of structurally related ABPs demonstrated different labeling efficiency and selectivity: the cyclooctyne-containing probes failed to label recombinant sirtuins to any appreciable level, while the azido-containing ABPs showed good isoform selectivity. The azido-containing ABPs were further analyzed for their ability to label an individual sirtuin isoform in protein mixtures and cell lysates. These biocompatible ABPs allow the study of dynamic cellular protein activity change to become possible.

Suppression of isoprenylcysteine carboxylmethyltransferase compromises DNA damage repair.

DNA damage is a double-edged sword for cancer cells. On the one hand, DNA damage-induced genomic instability contributes to cancer development; on the other hand, accumulating damage compromises proliferation and survival of cancer cells. Understanding the key regulators of DNA damage repair machinery would benefit the development of cancer therapies that induce DNA damage and apoptosis. In this study, we found that isoprenylcysteine carboxylmethyltransferase (ICMT), a posttranslational modification enzyme, plays an important role in DNA damage repair. We found that ICMT suppression consistently reduces the activity of MAPK signaling, which compromises the expression of key proteins in the DNA damage repair machinery. The ensuing accumulation of DNA damage leads to cell cycle arrest and apoptosis in multiple breast cancer cells. Interestingly, these observations are more pronounced in cells grown under anchorage-independent conditions or grown in vivo. Consistent with the negative impact on DNA repair, ICMT inhibition transforms the cancer cells into a "BRCA-like" state, hence sensitizing cancer cells to the treatment of PARP inhibitor and other DNA damage-inducing agents.

The RaPID Platform for the Discovery of Pseudo-Natural Macrocyclic Peptides.

Although macrocyclic peptides bearing exotic building blocks have proven their utility as pharmaceuticals, the sources of macrocyclic peptide drugs have been largely limited to mimetics of native peptides or natural product peptides. However, the recent emergence of technologies for discovering de novo bioactive peptides has led to their reconceptualization as a promising therapeutic modality. For the construction and screening of libraries of such macrocyclic peptides, our group has devised a platform to conduct affinity-based selection of massive libraries (>1012 unique sequences) of in vitro expressed macrocyclic peptides, which is referred to as the random nonstandard peptides integrated discovery (RaPID) system. The RaPID system integrates genetic code reprogramming using the FIT (flexible in vitro translation) system, which is largely facilitated by flexizymes (flexible tRNA-aminoacylating ribozymes), with mRNA display technology.We have demonstrated that the RaPID system enables rapid discovery of various de novo pseudo-natural peptide ligands for protein targets of interest. Many examples discussed in this Account prove that thioether-closed macrocyclic peptides (teMPs) obtained by the RaPID system generally exhibit remarkably high affinity and specificity, thereby potently inhibiting or activating a specific function(s) of the target. Moreover, such teMPs are used for a wide range of biochemical applications, for example, as crystallization chaperones for intractable transmembrane proteins and for in vivo recognition of specific cell types. Furthermore, recent studies demonstrate that some teMPs exhibit pharmacological activities in animal models and that even intracellular proteins can be inhibited by teMPs, illustrating the potential of this class of peptides as drug leads.Besides the ring-closing thioether linkage in the teMPs, genetic code reprogramming by the FIT system allows for incorporation of a variety of other exotic building blocks. For instance, diverse nonproteinogenic amino acids, hydroxy acids (ester linkage), amino carbothioic acid (thioamide linkage), and abiotic foldamer units have been successfully incorporated into ribosomally synthesized peptides. Despite such enormous successes in the conventional FIT system, multiple or consecutive incorporation of highly exotic amino acids, such as d- and β-amino acids, is yet challenging, and particularly the synthesis of peptides bearing non-carbonyl backbone structures remains a demanding task. To upgrade the RaPID system to the next generation, we have engaged in intensive manipulation of the FIT system to expand the structural diversity of peptides accessible by our in vitro biosynthesis strategy. Semilogical engineering of tRNA body sequences led to a new suppressor tRNA (tRNAPro1E2) capable of effectively recruiting translation factors, particularly EF-Tu and EF-P. The use of tRNAPro1E2 in the FIT system allows for not only single but also consecutive and multiple elongation of exotic amino acids, such as d-, β-, and γ-amino acids as well as aminobenzoic acids. Moreover, the integration of the FIT system with various chemical or enzymatic posttranslational modifications enables us to expand the range of accessible backbone structures to non-carbonyl moieties prominent in natural products and peptidomimetics. In such systems, FIT-expressed peptides undergo multistep backbone conversions in a one-pot manner to yield designer peptides composed of modified backbones such as azolines, azoles, and ring-closing pyridines. Our current research endeavors focus on applying such in vitro biosynthesis systems for the discovery of bioactive de novo pseudo-natural products.

Peptidylarginine deiminases 4 as a promising target in drug discovery

Peptidylarginine deaminase 4 (PAD4) is a crucial post-translational modifying enzyme catalyzing the conversion of arginine into citrulline residues, and mediating the formation of neutrophil extracellular traps (NETs). PAD4 plays a vital role in the occurrence and development of cardiovascular diseases, autoimmune diseases, and various tumors. Therefore, PAD4 is considered as a promising drug target for disease diagnosis and treatment. More and more efforts are devoted to developing highly efficient and selective PAD4 inhibitors via high-throughput screening, structure-based drug design and structure-activity relationship study. This article outlined the physiological and pathological functions of PAD4, and corresponding representative small molecule inhibitors reported in recent years.

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics.

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular level. MabThera® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.

Chemical Proteomics Approach for Profiling the NAD Interactome.

Nicotinamide adenine dinucleotide (NAD+) is a multifunctional molecule. Beyond redox metabolism, NAD+ has an equally important function as a substrate for post-translational modification enzymes, the largest family being the poly-ADP-ribose polymerases (PARPs, 17 family members in humans). The recent surprising discoveries of noncanonical NAD (NAD+/NADH)-binding proteins suggests that the NAD interactome is likely larger than previously thought; yet, broadly useful chemical tools for profiling and discovering NAD-binding proteins do not exist. Here, we describe the design, synthesis, and validation of clickable, photoaffinity labeling (PAL) probes, 2- and 6-ad-BAD, for interrogating the NAD interactome. We found that 2-ad-BAD efficiently labels PARPs in a UV-dependent manner. Chemical proteomics experiments with 2- and 6-ad-BAD identified known and unknown NAD+/NADH-binding proteins. Together, our study shows the utility of 2- and 6-ad-BAD as clickable PAL NAD probes.

Post-translational modification enzymes as key regulators of ciliary protein trafficking

Primary cilia are evolutionarily conserved microtubule-based organelles that protrude from the surface of almost all cell types and decode a variety of extracellular stimuli. Ciliary dysfunction causes human diseases named ciliopathies, which span a wide range of symptoms, such as developmental and sensory abnormalities. The assembly, disassembly, maintenance and function of cilia rely on protein transport systems including intraflagellar transport (IFT) and lipidated protein intraflagellar targeting (LIFT). IFT is coordinated by three multisubunit protein complexes with molecular motors along the ciliary axoneme, while LIFT is mediated by specific chaperones that directly recognize lipid chains. Recently, it has become clear that several post-translational modification enzymes play crucial roles in the regulation of IFT and LIFT. Here, we review our current understanding of the roles of these post-translational modification enzymes in the regulation of ciliary protein trafficking as well as their regulatory mechanisms, physiological significance and involvement in human diseases.

System-wide identification and prioritization of enzyme substrates by thermal analysis

Despite the immense importance of enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.

Role of Poly(ADP-ribose) Polymerase (PARP1) in Viral Infection and its Implication in SARS-CoV-2 Pathogenesis.

Activation of Poly (ADP-ribose) polymerase 1 (PARP1), a post-translational modifying enzyme, has been shown to be involved with several inflammatory and viral diseases. The goal of this review is to highlight the mechanisms underlying PARP1 activation during viral or infectious pathogenesis and to assess potential possibilities of using PARP1 inhibitors as a therapeutic countering of SARS-CoV-2 virus. An extensive bibliographic search was done using Pubmed, Mendeley and google scholar with key words. Pre-prints are reported with potential caveats and studies without experimental data were excluded. Covid-19, a global pandemic; is associated with systemic surge of inflammatory cytokines resulting in severe inflammation of the lung, heart dysfunction, ischemia, and stroke. PARP1 regulates expression of NFkB and downstream cytokine production and its inhibition is known to attenuate the expression of inflammatory cytokines. PARP1 and other PARP family members regulate viral infection, replication, and virulence. The literature clearly suggests that PARP1 plays an important role in host-pathogen interactions and pathogenesis, with pre-clinical and in vitro studies supporting the idea that PARP1 inhibition may negatively affect viability of several viruses including the replication of the SARS-CoV and SARS-CoV-2 virus. The current review discusses mechanisms of PARP1 activation during viral infection, inflammatory diseases, cytokine expression and possibility of PARP1 in regulating cytokine storm and hyper-inflammation seen with Covid-19. Additionally, in vitro studies showing the negative regulation of SARS-CoV-2 virus replication by PARP inhibitors indicates a potential therapeutic role of PARP inhibitors for Covid-19 or its variants.