Search Results (108)

Obesity and type 2 diabetes mellitus (T2DM) represent pandemic metabolic illnesses hallmarked by defective pancreatic β-cell function and blunted insulin release. As a conserved small GTPase (guanosine triphosphatase), ADP-ribosylation factor 6 (ARF6) governs fundamental cellular events encompassing vesicle trafficking, cytoskeleton remodeling and lipid metabolic turnover. Emerging data confirm that ARF6 acts as a master rheostat of glucose-stimulated insulin secretion (GSIS) in β-cells through downstream cell division control protein 42/Ras-related C3 botulinum toxin substrate 1 (Cdc42/Rac1) cascades. Pathogenic ARF6 hyperactivation triggers a cascade of β-cell lesions: mitochondrial impairment, autophagic suppression and exacerbated inflammatory signaling, accelerating the progression of obesity and T2DM. First-line therapeutics ranging from GLP-1 (Glucagon-like peptide-1) receptor agonists and metformin to SGLT2 (Sodium-Glucose Cotransporter 2) inhibitors partially restore metabolic homeostasis by rectifying aberrant ARF6-dependent signaling axes. This review comprehensively delineates ARF6’s canonical cellular roles, mechanistic bridges connecting ARF6 to β-cell failure and metabolic deterioration, and functional crosstalk between ARF6 and established anti-metabolic pharmacotherapies. We further address unresolved research gaps and prospective translational avenues, offering actionable perspectives to advance ARF6 as a tractable therapeutic target for obesity and T2DM management. Full article

Sclerotinia sclerotiorum is a formidable soilborne fungus that wreaks havoc on numerous crops globally. While the role of ADP-ribosylation factor-like 1 (Arl1) small GTPases in vesicular trafficking and fungal development is well-documented, their specific impact on S. sclerotiorum remains unclear. Through reverse genetic techniques, we identified and characterized SsArl1, a typical Arl small GTPase conserved across fungi. Deleting SsArl1 hampers the hyphal growth of S. sclerotiorum, but leads to higher oxalic acid buildup and boosts cellulase activity. This speeds up the infection of host plants, yet increases their sensitivity to certain environmental stresses, particularly ionic and cell wall-related stress. Our results reveal that SsArl1 acts as a negative regulator of oxalic acid accumulation and virulence, while playing a positive role in enhancing resistance to environmental stresses in S. sclerotiorum. Full article

The life-threatening disease pertussis, also known as whooping cough, is caused by a complex interplay of several virulence factors produced by the bacterium Bordetella (B.) pertussis. These include the AB-type protein toxin pertussis toxin (PT), the main causative agent of pertussis. After infection with B. pertussis, PT is released and binds to its human target cells, which internalize PT. The enzyme subunit of PT is then taken up into the cytosol, where it catalyzes the ADP-ribosylation of the α-subunit of inhibitory GTP-binding proteins from the Gαi type. This ultimately leads to the development of the characteristic clinical symptoms associated with pertussis. Pertussis is a vaccine-preventable but highly infectious respiratory disease, and especially younger children are prone to develop severe pertussis. Despite the vaccination, over the past few years, increasing case numbers have been reported globally. Moreover, treatment options are strongly limited to antibiotics and symptomatic treatment. Therefore, novel therapies against toxin-mediated diseases are urgently required, while AB-type toxins such as PT are promising pharmacological targets to combat these associated diseases. To identify novel pharmacological inhibitors for AB-type toxins, huge potential lies within the human proteome/peptidome. Endogenous protein or peptide inhibitors for bacterial toxins might have evolved as part of the innate immunity and are awaited to be discovered. The scientific community is committed to identify potential candidates through targeted screening or explorative hypothesis-driven approaches. This review summarizes the recent efforts in the identification and characterization of the human body’s own proteins and peptides that inhibit PT. PT-inhibiting peptides were found by unbiased screening of peptide libraries from human hemofiltrate or hypothesis-driven evaluation, and PT-neutralizing mechanisms were discovered in cell-based approaches. The identification of endogenous peptides and proteins, e.g., defensins and α₁-antitrypsin, as potent inhibitors of PT paves the way towards the development of novel therapeutic options against pertussis. Full article

Pertussis toxin (PTx) is a major virulence factor of Bordetella pertussis and an AB5-type exotoxin that disrupts host signaling. Its enzymatic A subunit ADP-ribosylates the α-subunit of inhibitory G proteins (Gα_i), preventing them from mediating receptor-induced inhibition of adenylyl cyclase (AC). This leads to unrestrained cAMP accumulation in host cells, a canonical mechanism underlying many pertussis disease manifestations. PTx works in concert with the bacterium’s adenylate cyclase toxin (ACT) to subvert immune defenses and establish infection. Interestingly, PTx exerts both cAMP-dependent and cAMP-independent effects. In addition to the well-known cAMP-mediated pathway, PTx’s B oligomer can engage host cell surface receptors to trigger signaling cascades independent of the A subunit’s catalytic activity. Such B oligomer-mediated pathways modulate cellular responses in the absence of ADP-ribosylation. This review provides a comprehensive analysis of PTx’s dual functionality, distinguishing its G_i protein-dependent elevation of cAMP from the noncanonical activities of the B oligomer. It also highlights how disruption of constitutive G_i signaling and the interplay between PTx and ACT shape host–pathogen interaction in pertussis pathogenesis. Full article

Tankyrases (TNKS1 and TNKS2) are multifunctional enzymes of the poly(ADP-ribose) polymerase (PARP) family that regulate cellular homeostasis by catalyzing poly(ADP-ribosyl)ation and stabilizing protein–protein interactions through their ankyrin repeat clusters. By engaging with diverse sets of proteins, TNKSs act as central hubs that coordinate signaling and metabolic pathways. In this review, we discuss how TNKS –protein interactions underpin their roles across multiple biological pathways, including Wnt/β-catenin, YAP and SRC signaling, mTORC1 signaling, DNA damage repair (via PARP crosstalk and recruitment of repair factors), telomere maintenance, cell-cycle regulation, glucose metabolism, cytoskeleton rearrangement, autophagy, proteasomal degradation, and apoptosis. We highlight the structural basis of these interactions, emphasizing ankyrin repeat domain recognition motifs and the consequences of TNKS-mediated PARylation on protein stability and localization. By integrating findings from oncology, virology, and metabolism, we illustrate how TNKS functions as a nodal regulator linking genome stability, signaling fidelity, and metabolic control. The interplay between TNKS and these varied pathways is essential for the well-being of the organism, with its dysregulation having severe biological and clinical consequences, which are discussed here. Finally, we consider therapeutic implications of disrupting TNKS–protein interactions, with particular attention paid to selective small-molecule inhibitors and their translational potential in cancer, viral infections, and degenerative diseases. Full article

Proteoglycans are macromolecules consisting of a core protein and one or more glycosaminoglycan side chains. Proteoglycans synthesized by vascular endothelial cells modulate various functions such as anticoagulant activity and vascular permeability. We previously reported that some heavy metals interfere with proteoglycan expression, and that organic–inorganic hybrid molecules, such as metal complexes and organometallic compounds, serve as useful tools to analyze proteoglycan synthesis mechanisms. However, the effects of metal compounds lacking electrophilicity on proteoglycan synthesis remain unclear. Au₂₅(SG)₁₈, a nanoscale gold cluster consisting of a metal core protected by gold–glutathione complexes, exhibits extremely low intramolecular polarity. In this study, we investigated the effect of Au₂₅(SG)₁₈ on proteoglycan synthesis in vascular endothelial cells. Au₂₅(SG)₁₈ accumulated significantly in vascular endothelial cells at low cell density and suppressed the expression of perlecan, a major heparan sulfate proteoglycan in cells, by inactivating ADP-ribosylation factor 6 (Arf6). Additionally, Au₂₅(SG)₁₈ reduced the expression of biglycan, a small dermatan sulfate proteoglycan, in vascular endothelial cells at low cell density; however, the underlying mechanisms remain unclear. Overall, our findings suggest that organic–inorganic hybrid molecules regulate the activity of Arf6-mediated protein transport to the extracellular space and that perlecan is regulated through this mechanism, highlighting the importance of Arf6-mediated extracellular transport for maintaining vascular homeostasis. Full article

ADP-ribosylation factor (Arf) proteins are small GTPases that regulate intracellular membrane trafficking and actin remodeling through tightly controlled cycles of GTP binding and hydrolysis. Arf1, a central coordinator of Golgi and endosomal transport, and Arf6, which regulates plasma membranes and endosomal dynamics, have both been implicated in late stages of the HIV-1 life cycle. However, the mechanisms by which these GTPases support viral assembly and release remain incompletely defined. Here, we provide direct evidence that both Arf1 and Arf6 are required for efficient trafficking of the HIV-1 Gag polyprotein, assembly, and virion production. Perturbation of Arf1 function using either GTP-locked (Q71L) or GDP-locked (T31N) mutants significantly reduced virus release, impaired Gag association with membrane compartments, and prevented its accumulation at the plasma membrane. Manipulation of Arf1 cycling through the GTPase-activating protein AGAP1 further demonstrated that dynamic transitions between GTP- and GDP-bound states are essential for productive Gag trafficking. Similarly, expression of a constitutively active Arf6 mutant (Q67L) misrouted Gag to intracellular membranes and markedly suppressed virion release. Importantly, disruption of Arf1 or Arf6 activity did not affect the expression, surface levels, or intracellular distribution of the host restriction factor BST-2. Together, these findings identify Arf1- and Arf6-mediated trafficking pathways as critical host determinants of HIV-1 assembly and release and establish that their functions operate independently of BST-2 antagonism. Full article

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by a lack of targetable receptors, leading to limited treatment options and a critical need for novel therapeutic strategies. This study aimed to evaluate the potential of azelastine, a clinically approved H1-antihistamine, for drug repositioning against TNBC and to elucidate its underlying HRH1-independent mechanism of action. Cell viability assays (CCK-8) were performed on TNBC cell lines (MDA-MB-231 and BT-549) following treatment with azelastine and its major metabolite, desmethyl azelastine. After observing ambiguous clinical associations between HRH1 expression and patient prognosis, HRH1 dependency was assessed through histamine stimulation and HRH1 knockdown (siRNA). Subsequently, the role of ADP-ribosylation factor 1 (ARF1), found to be overexpressed in TNBC and linked to poor prognosis, was investigated using ARF1 knockdown (siRNA), co-treatment with the Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) inhibitor golgicide A (GCA), and co-treatment with the Drp1 inhibitor M-divi 1. Azelastine and desmethyl azelastine potently reduced MDA-MB-231 cell viability in a dose- and time-dependent manner, achieving cell survivals of 61.3 ± 6.1% (30 µM) and 34.9 ± 3.7% (50 µM) for azelastine, and 52.4 ± 12.5% (30 µM) for desmethyl azelastine, respectively, after 72 h, with an IC₅₀ of 35.93 µM determined for azelastine in MDA-MB-231 cells. Additionally, azelastine significantly reduced the viability of BT-549 cells. Bioinformatic analysis of clinical datasets revealed HRH1 downregulation in tumors and, functionally, neither histamine stimulation nor HRH1 knockdown mediated azelastine cytotoxicity in cell culture. Importantly, ARF1 expression was significantly upregulated in TNBC and associated with poor prognosis. Co-treatment with GCA, preventing ARF1 activation, restored viability to near-control levels, supporting dependence on the GBF1–ARF1 activation axis of azelastine, whereas the Dynamic-related protein 1 (Drp1) inhibitor M-divi 1 not only partially rescued CCK-8-based cell viability but also normalized azelastine-induced loss of MitoTracker™ Red CMXRos signal and partially preserved (4′,6-diamidino-2-phenylindole) DAPI-based cell density, indicating Drp1-dependent mitochondrial dysfunction. Furthermore, azelastine selectively reduced p-ERK phosphorylation in the cell signaling pathway. Azelastine exerts potent anticancer effects in TNBC cells via an HRH1-independent, ARF1-dependent mechanism that attenuates the Extracellular signal-regulated kinase (ERK)–Drp1 axis, and induces Drp1-dependent mitochondrial dysfunction, independent of its canonical HRH1 receptor function. This ARF1-dependent mechanism provides strong scientific rationale for the drug repositioning of azelastine as an effective therapeutic agent for ARF1-driven TNBC. Full article

Sclerotinia sclerotiorum, a soil-borne phytopathogenic fungus with a broad host range, often leads to severe disease and significant economic losses in agricultural production. The guanine exchange factor EFA6 of ADP-ribosylation factor 6 (ARF6) has been extensively studied in animals, but its function in fungi is seldom reported. Here, reverse genetics methods were employed to explore the effects of SsEFA6 in the process of pathogenicity of S. sclerotiorum. Knockout of SsEFA6 hindered appressoria formation and sclerotia production. However, it did not affect the secretion of oxalic acid, the sensitivity to cell wall inhibitors, or hyperosmotic stress. Nevertheless, SsEFA6 deletion did result in a significant decrease in mutant virulence, indicative of its indispensability in virulence. Therefore, SsEFA6 plays an essential role in appressoria formation, sclerotia production, and fungal virulence in S. sclerotiorum. Full article

Poly(ADP-ribose)polymerase 1 (PARP1) is an enzyme that interacts with chromatin during DNA repair and transcription processes; the molecular mechanisms of these processes remain to be determined. Previously, we have shown that PARP1 can bind to and reorganize nucleosomes using two modes of interaction with a mono-nucleosome, which are realized through PARP1 binding to the ends of linker DNA and to the nucleosome core. Here, it is shown that the latter mode of binding induces the reorganization of nucleosome structure and is more stable under the conditions of poly(ADP-ribosyl)ation (PARylation). The initial nucleosome structure is fully recovered after the dissociation of autoPARylated PARP1. The competition between PARP1 and linker histone H1.0 for binding to a nucleosome is mediated by the PARP1-H1.0 interaction and is affected by the length of linker DNA fragments. Longer linkers stabilize H1.0-nucleosome complexes, while shorter linkers facilitate displacement of H1.0 from the chromatosome by PARP1. PARylation removes both H1.0 and PARP1 from the complexes with nucleosomes. The data suggest that the H1.0 displacement from chromatin by PARP1 that is likely modulated by the density of nucleosomes might reduce chromatin compaction and facilitate access of PARP1-dependent DNA repair and transcription factors to nucleosome and inter-nucleosomal DNA. Full article

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

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

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