Search Results (292)

Cardiomyocyte hypertrophy is regulated by several factors, including the ADP-ribosylation factor (Arf) family of small G proteins, among others. For instance, ArfGAP with dual pleckstrin homology domains 1 (Adap1) exerts an anti-hypertrophic effect in cultured cardiomyocytes. Its homologous protein, Adap2, is also expressed in the heart but its role remains elusive. To elucidate its function, we investigated the effects of adenoviral-mediated overexpression of Adap2 in cultured neonatal rat ventricular myocytes under both basal and pro-hypertrophic conditions, employing a range of microscopy and biochemical techniques. Despite minimal detection in neonatal rat hearts, Adap2 was found to be well expressed in adult rat hearts, being predominantly localized at the membrane fraction. In contrast to Adap1, overexpression of Adap2 provokes the robust accumulation of β1-integrin at the cellular surface of cultured cardiomyocytes. Interestingly, overexpressed Adap2 relocalizes at the sarcolemma and increases the size of cardiomyocytes upon phenylephrine stimulation, despite attenuating Erk1/2 phosphorylation and Nppa gene expression. Under these same conditions, cardiomyocytes overexpressing Adap2 also express higher level of detyrosinated tubulin, a marker of hypertrophic response. These findings provide new insights into the pro-hypertrophic function of Adap2 in cardiomyocytes. Full article

Phosphoinositide-binding pleckstrin homology (PH) domains interact with both phospholipids and proteins, often complicating their use as specific lipid biosensors. In this study, we introduced specific mutations into the phosphatidylinositol 3,4,5-trisphosphate (PIP₃)-specific PH domains of protein kinase B (Akt) and general receptor for phosphoinositides 1 (GRP1) that disrupt protein-mediated interactions while preserving lipid binding, in order to enhance biosensor specificity for PIP₃, and evaluated their impact on plasma membrane (PM) localization and lipid-tracking ability. Using bioluminescence resonance energy transfer (BRET) and confocal microscopy, we assessed the localization of PH domains in HEK293A cells under different conditions. While Akt-PH mutants showed minimal deviations from the wild type, GRP1-PH mutants exhibited significantly reduced PM localization both at baseline and after stimulation with epidermal growth factor (EGF), insulin, or vanadate. We further developed tandem mutant GRP1-PH domain constructs to enhance PM PIP₃ avidity. Additionally, our investigation into the influence of ADP ribosylation factor 6 (Arf6) activity on GRP1-PH-based biosensors revealed that while the wild-type sensors were Arf6- dependent, the mutants operated independently of Arf6 activity level. These optimized GRP1-PH constructs provide a refined biosensor system for accurate and selective detection of dynamic PIP₃ signaling, expanding the toolkit for dissecting phosphoinositide-mediated pathways. Full article

Fruit yield and quality improvement are challenges for researchers and farmers. This study reveals that the main fruit traits of blueberry (Vaccinium ashei ‘Bluegem’) were significantly improved after hydrogen (H₂)-based irrigation, assessed by the increased single fruit weight (14.59 ± 6.66%) and fruit equatorial diameter (4.19 ± 2.39%), decreased titratable acidity, increased solid–acid and sugar–acid ratios. The enhancement of fruit quality was confirmed by the increased total volatiles, vitamin C contents, and antioxidant capacity. Using weighted protein co-expression network analysis (WPCNA), proteomic interrogation revealed that serine carboxypeptidase-like proteins I/II (SCPLI/II), ADP ribosylation factor 1/2 (ARF1/2), and UDP-glucosyltransferase 85A (UGT85A) might be functionally associated with the increased fruit weight and size driven by H₂. Reduced organic acid accumulation was caused by the regulation of the specific enzymes involved in sucrose metabolism (e.g., α-amylase, endoglucanase, β-glucosidase, etc.). H₂ regulation of fatty acid degradation (e.g., acyl CoA oxidase 1 (ACX1), acetyl CoA acyltransferase 1 (ACAA1), etc.) and phenylpropanoid metabolism were used to explain the improved fruit aroma and anthocyanin accumulation. Meanwhile, the upregulated heat shock protein 20/70 matched with the enhanced antioxidant activity. Together, this study provides a novel approach for yield and quality improvement in horticultural crops. Full article

Poly(ADP-ribosyl)ation is a crucial posttranslational modification that governs gene expression, chromatin remodeling, and cellular homeostasis. This dynamic process is mediated by the opposing activities of poly(ADP-ribose) polymerases (PARPs), which synthesize poly(ADP-ribose) (pADPr), and poly(ADP-ribose) glycohydrolase (PARG), which degrades it. While PARP function has been extensively studied, the structural and mechanistic basis of PARG-mediated pADPr degradation remain incompletely understood. To investigate the role of PARG in pADPr metabolism, we employed CRISPR/Cas9-based genome editing to generate a novel Parg^29b mutant mouse embryonic stem cell (ESC) line carrying a precise deletion within the PARG catalytic domain. This deletion completely abolished pADPr hydrolytic activity, resulting in massive nuclear pADPr accumulation, yet ESC viability, proliferation, and cell cycle progression remained unaffected. Using Drosophila melanogaster as a model system, we demonstrated that this mutation completely disrupted the pADPr pathway and halted developmental progression, highlighting the essential role of PARG and pADPr turnover in organismal development. Our results define a critical structural determinant of PARG catalytic function, underscore the distinct requirements for pADPr metabolism in cellular versus developmental contexts, and provide a genetically tractable model for studying the regulation of poly(ADP-ribose) dynamics and therapeutic responses to PARP inhibition in vivo. Full article

Background/Objectives: Motile aeromonads are ubiquitous aquatic Gram-negative opportunistic pathogens with environmental, animal, aquatic, and human health implications. Methods: Motile aeromonads were isolated from village pond water samples (n = 100) of the Hisar district of Haryana state in India. Selective isolation and enumeration were followed by biochemical and genotypic identification using gyrB gene; evaluation of seven putative virulence factors and antimicrobial resistance studies and determination of extended spectrum beta lactamase (ESBL) and AmpC beta lactamase (ACBL) enzyme-producing abilities took place. Results: The viable counts of motile aeromonads varied from 1.6 × 10² CFU/mL to 1.2 × 10⁸ CFU/mL. Six species of Aeromonas were identified with high prevalence of A. veronii (74.7%), followed by A. caviae (8.9%), A. hydrophila (7.6), A. jandaei (5%), A. sobria (2.5%), and A. dhakensis (1.3%). PCR amplification of seven genes related to virulence indicated that the majority of the isolates were positive for enolase (eno, 98%), cytotoxic enterotoxin (act, 88%), and hemolysin (asa1, 86%). Many isolates were also positive for type III secretion system inner membrane component (ascV, 53%), ADP-ribosylating toxin (aexT, 47%), and extracellular hemolysin (ahh1, 4%). The antimicrobial resistance (AMR) profile of the isolated Aeromonas isolates indicated the high resistance observed to nalidixic acid (40.2%), cefoxitin (33%), and imipenem (6.2%). In addition, the occurrence of 10.3% ESBL, 32% ACBL, and 29.9% multi-drug resistant (MDR) isolates is alarming. Phylogenetic analysis of gyrB sequences of A. veronii isolates (n = 59) together with GenBank sequences of A. veronii from different geographical regions of the world indicated high genotypic diversity. Conclusions: the village aquaculture ponds in Hisar district have a high occurrence of MDR A. veronii, A. hydrophila, and A. caviae, posing significant animal and public health concern. Full article

The maintenance of genome stability and the prevention of genotoxic damage to DNA require immediate DNA repair. In the cell, the repair process is usually preceded by a release of DNA from complexes with chromatin proteins accompanied by nucleosome sliding, relaxing or disassembly. Base excision DNA repair (BER) corrects the most common DNA lesions, which does not disturb the DNA helix dramatically. Notably, small DNA lesions can be repaired in chromatin without global chromatin decompaction. One of the regulatory mechanisms is poly(ADP-ribosyl)ation, leading to the relaxation of the nucleosome. In our work, we demonstrated that recently a discovered protein, HPF1, can modulate the efficiency of one of the key BER stages—DNA synthesis—via the regulation of total poly(ADP-ribosyl)ation. Accordingly, we investigated both short-patch and long-patch DNA synthesis catalyzed by DNA polymerase β (pol β; main polymerase in BER) and showed that HPF1’s influence on the poly(ADP-ribosyl)ation catalyzed by PARP1 and especially by PARP2 results in more efficient DNA synthesis in the case of the short-patch BER pathway in nucleosomes. Additionally, HPF1-dependent poly(ADP-ribosyl)ation was found to positively regulate long-patch BER. Full article

Magnesium, the most abundant divalent metal within the cell, is essential for physiological function and critical in cellular signaling. To maintain cellular homeostasis, intracellular magnesium levels are tightly regulated, as dysregulation is linked to numerous diseases, including cancer, diabetes, cardiovascular disorders, and neurological conditions. Over the past two decades, extensive research on magnesium-regulating proteins has provided valuable insight into their pathogenic and therapeutic potential. This review explores an emerging mechanism of magnesium homeostasis involving proteins in the PRL (phosphatase of regenerating liver), ARL (ADP ribosylation factor-like GTPase family), CNNM (cyclin and cystathionine β-synthase domain magnesium transport mediator), and TRPM (transient receptor potential melastatin) families, collectively termed herein as the PACT network. While each PACT protein has been studied within its individual signaling and disease contexts, their interactions suggest a broader regulatory network with therapeutic potential. This review consolidates the current knowledge on the PACT proteins’ structure, function, and interactions and identifies research gaps to encourage future investigation. As the field of magnesium homeostasis continues to advance, understanding PACT protein interactions offers new opportunities for basic research and therapeutic development targeting magnesium-related disorders. Full article

Poly(ADP-ribosyl)ation (PARylation) is a dynamic protein post-translational modification (PTM) mediated by ADP-ribosyltransferases (ARTs), which regulates a plethora of essential biological processes, such as DNA repair, gene expression, and signal transduction. Among these, PAR-dependent ubiquitination (PARdU) plays a pivotal role in tagging PARylated substrates for subsequent ubiquitination and degradation events through the coordinated action of enzymes, including the E3 ligase RNF146 and the ADP-ribosyltransferase tankyrase. Notably, this pathway has emerged as a key regulator of tumorigenesis, immune modulation, and cell death. This review elucidates the molecular mechanisms of the PARdU pathway, including the RNF146–tankyrase interaction, substrate specificity, and upstream regulatory pathways. It also highlights the biological functions of PARdU in DNA damage repair, signaling pathways, and metabolic regulation, with a focus on its therapeutic potential in cancer treatment. Strategies targeting PARdU, such as tankyrase and RNF146 inhibitors, synthetic lethality approaches, and immune checkpoint regulation, offer promising avenues for precision oncology. These developments underscore the potential of PARdU as a transformative therapeutic target in combating various types of human cancer. Full article

Balamuthia mandrillaris is a free-living amoeba pathogenic to humans, causing amoebic granulomatous encephalitis (GAE). Due to the associated mortality rates of <95%, the absence of treatments, and a clear understanding of the pathogenesis of this amoeba, Lippia graveolens could be an interesting alternative since it has been used against bacteria, fungi, and other pathogenic protozoa. This study employed RNA sequencing to analyze differentially expressed genes (DEGs), following treatment with two fractionated L. graveolens extracts (concentration: 150 µg/mL) at 48, 96, and 120 h. The DEGs identified are associated with several functions such as stress responses (Prohibitin domain-containing protein), and oxidative damage repair and cell stability (Peroxiredoxin). Genes implicated in virulence and host interaction also showed significant expression changes, such as the ADP ribosylation factor (Arf) GTPase and ephrin type-A receptor, alongside transcription factors involved in the phagocytosis of amoebas. Additionally, the analysis of Gene Ontology categories revealed terms including transmembrane signaling receptor and protein tyrosine activity, DNA replication initiation, the mitotic M phase, and membrane integrity. These results provide valuable insights into the molecular mechanisms utilized by B. mandrillaris to respond to environmental stressors and the repression of genes related to essential functions, which could serve as potential targets for developing novel strategies. Full article

The role of the chikungunya virus (CHIKV) non-structural protein 3 (nsP3) in the virus lifecycle is poorly understood. The protein comprises three domains. At the N-terminus is a macro domain, biochemically characterised to bind both RNA and ADP-ribose and to possess ADP-ribosyl hydrolase activity—an enzymatic activity that removes ADP-ribose from mono-ADP-ribosylated proteins. As ADP-ribosylation is important in the signalling pathway, leading to activation of the transcription factor NF-κB, we sought to determine whether the macro domain might perturb NF-κB signalling. We first showed that CHIKV infection did not induce NF-κB activation and could not block exogenous activation of the pathway via TNFα, although TNFα treatment did result in a modest reduction in virus titre. In contrast, ectopic expression of nsP3 was able to inhibit both basal and TNFα-mediated NF-κB activation, and this was dependent on the macro domain, as a mutation previously shown to disrupt ADP-ribose binding and hydrolase activity (D10A) eliminated the ability to inhibit NF-κB activation. The macro domain D10A mutant also resulted in a dramatic reduction in virus infectivity, consistent with the notion that the ability of the macro domain to inhibit NF-κB activation plays a role in the virus lifecycle. Full article

ADP-ribosylation is a reversible modification of proteins and nucleic acids, which controls major cellular processes, including DNA damage repair, cell proliferation and differentiation, metabolism, stress, and immunity in plants and animals. The involvement of ADP-ribosylation in the life cycle of Dictyostelium and some filamentous fungi has also been demonstrated. However, the role of this process in pathogenic oomycetes has never been addressed. Here, we show that the Phytophthora infestans genome contains two PARP-like protein genes (PiPARP1 and PiPARP2), and provide evidence of PARylation activity for one of them (PiPARP2). Using dsRNA-mediated RNA silencing of the PiPARP2 gene and chemical (pharmacological) inhibition of PARP activity by 3-aminobenzamide (3AB) PARP inhibitor, we demonstrate the critical functional role of ADP-ribosylation in Phytophthora mycelium growth. Virulence test on detached leaves also suggests an important role of ADP-ribosylation in Phytophthora host plant colonisation and pathogenesis. On a practical level, our data suggest that targeting the PARylation system may constitute a novel powerful approach for the management of Phytophthora diseases. Full article

The mammalian Apolipoprotein-L families (APOLs) contain several isoforms of membrane-interacting proteins, some of which are involved in the control of membrane dynamics (traffic, fission and fusion). Specifically, human APOL1 and APOL3 appear to control membrane remodeling linked to pathogen infection. Through its association with Non-Muscular Myosin-2A (NM2A), APOL1 controls Golgi-derived trafficking of vesicles carrying the lipid scramblase Autophagy-9A (ATG9A). These vesicles deliver APOL3 together with phosphatidylinositol-4-kinase-B (PI4KB) and activated Stimulator of Interferon Genes (STING) to mitochondrion–endoplasmic reticulum (ER) contact sites (MERCSs) for the induction and completion of mitophagy and apoptosis. Through direct interactions with PI4KB and PI4KB activity controllers (Neuronal Calcium Sensor-1, or NCS1, Calneuron-1, or CALN1, and ADP-Ribosylation Factor-1, or ARF1), APOL3 controls PI(4)P synthesis. PI(4)P is required for different processes linked to infection-induced inflammation: (i) STING activation at the Golgi and subsequent lysosomal degradation for inflammation termination; (ii) mitochondrion fission at MERCSs for induction of mitophagy and apoptosis; and (iii) phagolysosome formation for antigen processing. In addition, APOL3 governs mitophagosome fusion with endolysosomes for mitophagy completion, and the APOL3-like murine APOL7C is involved in phagosome permeabilization linked to antigen cross-presentation in dendritic cells. Similarly, APOL3 can induce the fusion of intracellular bacterial membranes, and a role in membrane fusion can also be proposed for endothelial APOLd1 and adipocyte mAPOL6, which promote angiogenesis and adipogenesis, respectively, under inflammatory conditions. Thus, different APOL isoforms play distinct roles in membrane remodeling associated with inflammation. Full article

Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) play a key role in DNA repair. As major sensors of DNA damage, they are activated to produce poly(ADP-ribose). PARP1/PARP2 inhibitors have emerged as effective drugs for the treatment of cancers with BRCA deficiencies. Here, we explored aminomethylmorpholino and aminomethylmorpholino glycine nucleosides as inhibitors of PARP1 and PARP2, using different enzymatic assays. The compounds bearing thymine or 5-Br(I)-uracil bases displayed the highest inhibition potency, with all of them being more selective toward PARP1. Interaction of the inhibitors with the NAD⁺ binding cavity of PARP1 (PARP2) suggested by the mixed-type inhibition was demonstrated by molecular docking and the RoseTTAFold All-Atom AI-model. The best PARP1 inhibitors characterized by the inhibition constants in the range of 12–15 µM potentiate the cytotoxicity of hydrogen peroxide by displaying strong synergism. The inhibitors revealed no impact on PARP1/PARP2 affinity for DNA, while they reduced the dissociation rate of the enzyme–DNA complex upon the autopoly(ADP-ribosyl)ation reaction, thus providing evidence that their mechanism of action for PARP trapping is due primarily to catalytic inhibition. The most active compounds were shown to retain selectivity toward PARP1, despite the reduced inhibition potency in the presence of histone PARylation factor 1 (HPF1) capable of regulating PARP1/PARP2 catalytic activity and ADP-ribosylation reaction specificity. The inhibitors obtained seem to be promising for further research as potential drugs. Full article

Fused in sarcoma (FUS) is involved in the formation of nuclear biomolecular condensates associated with poly(ADP-ribose) [PAR] synthesis catalyzed by a DNA damage sensor such as PARP1. Here, we studied FUS microphase separation induced by poly(ADP-ribosyl)ated PARP1^WT [PAR-PARP1^WT] or its catalytic variants PARP1^Y986S and PARP1^Y986H, respectively, synthesizing (short PAR)-PARP1^Y986S or (short hyperbranched PAR)-PARP1^Y986H using dynamic light scattering, fluorescence microscopy, turbidity assays, and atomic force microscopy. We observed that biologically relevant cations such as Mg²⁺, Ca²⁺, or Mn²⁺ or polyamines (spermine⁴⁺ or spermidine³⁺) were essential for the assembly of FUS with PAR-PARP1^WT and FUS with PAR-PARP1^Y986S in vitro. We estimated the range of the FUS-to-PAR-PARP1 molar ratio and the cation concentration that are favorable for the stability of the protein’s microphase-separated state. We also found that FUS microphase separation induced by PAR-PARP1^Y986H (i.e., a PARP1 variant attaching short hyperbranched PAR to itself) can occur in the absence of cations. The dependence of PAR-PARP1-induced FUS microphase separation on cations and on the branching of the PAR structure points to a potential role of the latter in the regulation of the formation of FUS-related biological condensates and requires further investigation. Full article

Post-translational modifications (PTMs) are increasingly recognized as important strategies used by bacterial pathogens to modulate host cellular functions. Protein ADP-riboxanation, a derivative of ADP-ribosylation, has recently emerged as a new biochemical way by which bacterial pathogens interact with host cells. Recent studies have revealed that this modification has broad regulatory roles in host processes including cell death, protein translation, and stress granule formation. Given that the vast majority of bacterial ADP-riboxanases are still uncharacterized, in this review we also highlight the utility of advanced proteomic tools in the functional dissection of ADP-riboxanation events during bacterial infections. Full article