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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.

Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry. PARG and ARH3 are the main hydrolases to reverse serine poly(ADP-ribosylation) yet their activities in the process differ. Here, the authors synthesise linear and branched poly(ADP-ribose) molecules, perform structure-function analysis and elucidate the mechanistic differences between PARG and ARH3.

Mono(ADP-ribosyl)ation (MARylation) is a regulatory post-translational modification of proteins that controls their functions through a variety of mechanisms. MARylation is catalyzed by mono(ADP-ribosyl) transferase (MART) enzymes, a subclass of the poly(ADP-ribosyl) polymerase (PARP) family of enzymes. Although the role of PARPs and poly(ADP-ribosyl)ation (PARylation) in cellular pathways, such as DNA repair and transcription, is well studied, the role of MARylation and MARTs (i.e., the PARP ‘monoenzymes’) are not well understood. Moreover, compared to PARPs, the development of MART-targeted therapeutics is in its infancy. Recent studies are beginning to shed light on the structural features, catalytic targets, and biological functions of MARTs. The development of new technologies to study MARTs have uncovered essential roles for these enzymes in the regulation of cellular processes, such as RNA metabolism, cellular transport, focal adhesion, and stress responses. These insights have increased our understanding of the biological functions of MARTs in cancers, neuronal development, and immune responses. Furthermore, several novel inhibitors of MARTs have been developed and are nearing clinical utility. In this review, we summarize the biological functions and molecular mechanisms of MARTs and MARylation, as well as recent advances in technology that have enabled detection and inhibition of their activity. We emphasize PARP-7, which is at the forefront of the MART subfamily with respect to understanding its biological roles and the development of therapeutically useful inhibitors. Collectively, the available studies reveal a growing understanding of the biochemistry, chemical biology, physiology, and pathology of MARTs.

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD + substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3’ phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD + and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution. The mechanism of PARP1-dependent poly-ADP-ribosylation in response to DNA damage is still under debate. Here, the authors use ATR-FTIR spectroscopy to provide time-resolved insights into the molecular details of this process under near physiological conditions.

Background: The ADP‐ribosylation factor 1 (ARF1) gene encodes a protein which plays a critical role in intra‐Golgi transport. Clinical evidence suggests that individuals harbouring variants in the ARF1 gene display a consistent set of phenotypic features, including intellectual disability, microcephaly, epilepsy, and periventricular nodular heterotopia (PVNH). Methods: This study describes the case of a 6‐year and 5‐month‐old female presenting with focal seizures on a fixed side that were resistant to various anti‐seizure medications. The genetic aetiology was elucidated through exome sequencing of the pedigree. The pathogenicity of the identified variant was subsequently assessed using molecular dynamics structural analysis, western blotting, and co‐immunoprecipitation techniques. Results: A de novo variant, c.509T > C (p.Leu170Pro), was detected in the ARF1 gene, and functional analysis demonstrated that this modification is anticipated to hinder its association with the Golgi‐localising, γ‐adaptin ear homology domain and ARF‐binding protein, thereby playing a role in the pathogenesis of the disease. Conclusion: This study introduces the initial instance of ARF1‐related disease in China, wherein the patient is without the presence of PVNH. The findings add novel clinical phenotypes to the range of ARF1‐related diseases, and an investigation into the potential pathogenic mechanisms of this condition was conducted by confirming the deleterious impacts of the variant.

Protein ADP-ribosylation is essential for the regulation of several cellular pathways, enabling dynamic responses to diverse pathophysiological conditions. It is modulated through a dynamic interplay between ADP-ribose readers, writers and erasers. While ADP-ribose synthesis has been studied and reviewed extensively, ADP-ribose processing by erasing enzymes has received comparably less attention. However, major progress in the mass spectrometric identification of ADP-ribosylated residues and the biochemical characterization of ADP-ribose erasers has substantially expanded our knowledge of ADP-ribosylation dynamics. Herein, we describe recent insights into the biology of ADP-ribose erasers and discuss the intricately orchestrated cellular processes to switch off ADP-ribose-dependent mechanisms. ADP-ribose erasing enzymes are increasingly recognized as critical regulators of protein ADP-ribosylation dynamics in living systems. Here, the authors review recent advances in the discovery and characterization of ADP-ribose erasers and discuss their role within the cellular ADP-ribosylation machinery.

Various intracellular pathogens attempt to hide from innate cytosolic sensors by forming vacuoles. Yamamoto and colleagues show that the autophagy-related protein Gate-16, which is induced by interferon-γ, is required for noncanonical autophagy to control infection by Toxoplasma gondii . Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma . Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.

Lysine fatty acylation in mammalian cells was discovered nearly three decades ago, yet the enzymes catalyzing it remain unknown. Unexpectedly, we find that human N-terminal glycine myristoyltransferases (NMT) 1 and 2 can efficiently myristoylate specific lysine residues. They modify ADP-ribosylation factor 6 (ARF6) on lysine 3 allowing it to remain on membranes during the GTPase cycle. We demonstrate that the NAD + -dependent deacylase SIRT2 removes the myristoyl group, and our evidence suggests that NMT prefers the GTP-bound while SIRT2 prefers the GDP-bound ARF6. This allows the lysine myrisotylation-demyristoylation cycle to couple to and promote the GTPase cycle of ARF6. Our study provides an explanation for the puzzling dissimilarity of ARF6 to other ARFs and suggests the existence of other substrates regulated by this previously unknown function of NMT. Furthermore, we identified a NMT/SIRT2-ARF6 regulatory axis, which may offer new ways to treat human diseases. Lysine fatty acylation is an important protein posttranslational modification but mammalian lysine fatty acyl transferases have remained unknown so far. Here the authors report that the human N-terminal glycine myristoyltransferases 1 and 2 catalyze the addition of myristoyl chains to specific lysine residues and show that they myristoylate ARF6 lysine 3, which explains the unusual membrane binding properties of ARF6.

The adhesion receptor vascular endothelial (VE)-cadherin transduces an array of signals that modulate crucial lymphatic cell behaviors including permeability and cytoskeletal remodeling. Consequently, VE-cadherin must interact with a multitude of intracellular proteins to exert these functions. Yet, the full protein interactome of VE-cadherin in endothelial cells remains a mystery. Here, we use proximity proteomics to illuminate how the VE-cadherin interactome changes during junctional reorganization from dis-continuous to continuous junctions, triggered by the lymphangiogenic factor adrenomedullin. These analyses identified interactors that reveal roles for ADP ribosylation factor 6 (ARF6) and the exocyst complex in VE-cadherin trafficking and recycling. We also identify a requisite role for VE-cadherin in the in vitro and in vivo control of secretion of reelin—a lymphangiocrine glycoprotein with recently appreciated roles in governing heart development and injury repair. This VE-cadherin protein interactome shines light on mechanisms that control adherens junction remodeling and secretion from lymphatic endothelial cells. VE-cadherin is a cell adhesion and signaling molecule that regulates lymphatic cell behaviors. Here the authors show how the VE-cadherin interactome changes in response to junctional remodeling by adrenomedullin and identify mechanisms of VE-cadherin recycling and reelin secretion.

ADP-ribosylation is a posttranslational modification that exists in monomeric and polymeric forms. Whereas the writers (e.g. ARTD1/PARP1) and erasers (e.g. PARG, ARH3) of poly-ADP-ribosylation (PARylation) are relatively well described, the enzymes involved in mono-ADP-ribosylation (MARylation) have been less well investigated. While erasers for the MARylation of glutamate/aspartate and arginine have been identified, the respective enzymes with specificity for serine were missing. Here we report that, in vitro, ARH3 specifically binds and demodifies proteins and peptides that are MARylated. Molecular modeling and site-directed mutagenesis of ARH3 revealed that numerous residues are critical for both the mono- and the poly-ADP-ribosylhydrolase activity of ARH3. Notably, a mass spectrometric approach showed that ARH3-deficient mouse embryonic fibroblasts are characterized by a specific increase in serine-ADP-ribosylation in vivo under untreated conditions as well as following hydrogen peroxide stress. Together, our results establish ARH3 as a serine mono-ADP-ribosylhydrolase and as an important regulator of the basal and stress-induced ADP-ribosylome. Protein ADP-ribosylation has emerged as a key post translational modification that regulates several stress responses. Here the authors characterize ARH3 as a major serine-specific mono–ADP-­ribosylhydrolase and use a proteomics approach to identify the cellular targets of ARH3.