Plasma Membrane Reorganization Research Articles

Arf6 as a therapeutic target: Structure, mechanism, and inhibitors.

ADP-ribosylation factor 6 (Arf6), a small G-protein of the Ras superfamily, plays pivotal roles in multiple cellular events, including exocytosis, endocytosis, actin remodeling, plasma membrane reorganization and vesicular transport. Arf6 regulates the progression of cancer through the activation of cell motility and invasion. Aberrant Arf6 activation is a potential therapeutic target. This review aims to understand the comprehensive function of Arf6 for future cancer therapy. The Arf6 GEFs, protein structure, and roles in cancer have been summarized. Comprehending the mechanism underlying Arf6-mediated cancer cell growth and survival is essential. The structural features of Arf6 and its efforts are discussed and may be contributed to the discovery of future novel protein-protein interaction inhibitors. In addition, Arf6 inhibitors and mechanism of action are listed in the table. This review further emphasizes the crucial roles in drug resistance and attempts to offer an outlook of Arf6 in cancer therapy.

TBC1D10C is a cytoskeletal functional linker that modulates cell spreading and phagocytosis in macrophages

Cell spreading and phagocytosis are notably regulated by small GTPases and GAP proteins. TBC1D10C is a dual inhibitory protein with GAP activity. In immune cells, TBC1D10C is one of the elements regulating lymphocyte activation. However, its specific role in macrophages remains unknown. Here, we show that TBC1D10C engages in functions dependent on the cytoskeleton and plasma membrane reorganization. Using ex vivo and in vitro assays, we found that elimination and overexpression of TBC1D10C modified the cytoskeletal architecture of macrophages by decreasing and increasing the spreading ability of these cells, respectively. In addition, TBC1D10C overexpression contributed to higher phagocytic activity against Burkholderia cenocepacia and to increased cell membrane tension. Furthermore, by performing in vitro and in silico analyses, we identified 27 TBC1D10C-interacting proteins, some of which were functionally classified as protein complexes involved in cytoskeletal dynamics. Interestingly, we identified one unreported TBC1D10C-intrinsically disordered region (IDR) with biological potential at the cytoskeleton level. Our results demonstrate that TBC1D10C shapes macrophage activity by inducing reorganization of the cytoskeleton-plasma membrane in cell spreading and phagocytosis. We anticipate our results will be the basis for further studies focused on TBC1D10C. For example, the specific molecular mechanism in Burkholderia cenocepacia phagocytosis and functional analysis of TBC1D10C-IDR are needed to further understand its role in health and disease.

The Regulatory Role of GBF1 on Osteoclast Activation Through EIF2a Mediated ER Stress and Novel Marker FAM129A Induction.

Bone-resorbing activities of osteoclasts (OCs) are highly dependent on actin cytoskeleton remodeling, plasma membrane reorganization, and vesicle trafficking pathways, which are partially regulated by ARF-GTPases. In the present study, the functional roles of Golgi brefeldin A resistance factor 1 (GBF1) are proposed. GBF1 is responsible for the activation of the ARFs family and vesicular transport at the endoplasmic reticulum–Golgi interface in different stages of OCs differentiation. In the early stage, GBF1 deficiency impaired OCs differentiation and was accompanied with OCs swelling and reduced formation of mature OCs, indicating that GBF1 participates in osteoclastogenesis. Using siRNA and the specific inhibitor GCA for GBF1 knockdown upregulated endoplasmic reticulum stress-associated signaling molecules, including BiP, p-PERK, p-EIF2α, and FAM129A, and promoted autophagic Beclin1, Atg7, p62, and LC3 axis, leading to apoptosis of OCs. The present data suggest that, by blocking COPI-mediated vesicular trafficking, GBF1 inhibition caused intense stress to the endoplasmic reticulum and excessive autophagy, eventually resulting in the apoptosis of mature OCs and impaired bone resorption function.

Beyond pore formation: reorganization of the plasma membrane induced by pore-forming proteins.

Pore-forming proteins (PFPs) are a heterogeneous group of proteins that are expressed and secreted by a wide range of organisms. PFPs are produced as soluble monomers that bind to a receptor molecule in the host cell membrane. They then assemble into oligomers that are incorporated into the lipid membrane to form transmembrane pores. Such pore formation alters the permeability of the plasma membrane and is one of the most common mechanisms used by PFPs to destroy target cells. Interestingly, PFPs can also indirectly manipulate diverse cellular functions. In recent years, increasing evidence indicates that the interaction of PFPs with lipid membranes is not only limited to pore-induced membrane permeabilization but is also strongly associated with extensive plasma membrane reorganization. This includes lateral rearrangement and deformation of the lipid membrane, which can lead to the disruption of target cell function and finally death. Conversely, these modifications also constitute an essential component of the membrane repair system that protects cells from the lethal consequences of pore formation. Here, we provide an overview of the current knowledge on the changes in lipid membrane organization caused by PFPs from different organisms.

Functional implications of Pacsin2 localization in mast cells

Mast cell activation and degranulation involves rapid and dramatic reorganization of plasma membrane and cytoskeleton. We discovered that the protein Pacsin2 (syndapin II) localizes in distinct ring-like structures on resting mast cell plasma membranes. These rings surround areas of high F-actin and plasma membrane signals, resembling reservoirs. Activation through FCER1 or thapsigargin rapidly disassemble Pacsin2 rings and reservoirs, which reform when activation ceases. Localization of Pacsin2 to rings is dependent on the F-BAR domain region of the protein, which is responsible for plasma membrane bending and curvature sensing. Pacsin2 ring formation is anti-correlated with the activity of N-WASP, an Arp2/3-activating protein that promotes F-actin assembly and degranulation downstream of FCER1. Notably, N-WASP inhibition increases Pacsin2 localization at the plasma membrane. Due to its dynamic ring-like organization at membrane reservoirs and opposing relationship to N-WASP during cell activation, Pacsin2 may serve as a conduit between structural organization of the plasma membrane and downstream signaling to impact mast cell functions such as degranulation.

Role of neuroendocrine modulation and biochemistry in the sepsis in Piaractus mesopotamicus

Sepsis is a systemic process with multifactorial pathophysiology that affects most animal species. It is responsible for high rates of morbidity and mortality. This work aimed to study the biochemical and neuroendocrine changes of the sepsis process in Piaractus mesopotamicus after Aeromonas hydrophila inoculation analyzing changes in blood leukocyte and differences in neuroendocrine-biochemical modulation using RNA-seq. Fish showed hypercortisolemia, inhibition of glucose absorption, followed by hypocortisolemia and then hyperglycemia. Thyroid hormones (T3 and T4) showed immediate decrease in serum and T4 increased 6 h post-inoculation (HPI). Sepsis-induced hormonal alterations triggered changes in the metabolic pathways increasing protein and lipid catabolism, use of transient anaerobic glycolysis and liver injury. A reference transcriptome was constructed based on blood leukocytes from P. mesopotamicus. The assembly resulted in total 266,272 contigs with a N50 of 2786 bp. There was a reorganization of plasma membrane of leukocytes at the beginning of the septic process with increased expression of neuroendocrine receptors and with continuous flow of neurotransmitters, hormones and solutes with compensatory regulation at 6 HPI. Three and nine HPI seemed to be critical, the expression of a number of transcription factors was increased, including the modulatory DEGs related to glucocorticoid and thyroid hormones induced and suppressed (FDR < 0.05). Neuroendocrine modulation can regulate leukocytes and biochemical parameters of peripheral blood, being important sources for the study of the pathophysiology of sepsis. These finding highlights the importance of further studies focusing on biochemical-neuroendocrine changes in blood leukocytes and systemic sepsis.

GRK Mediates μ-Opioid Receptor Plasma Membrane Reorganization.

Differential regulation of the μ-opioid receptor (MOP) has been linked to the development of opioid tolerance and dependence which both limit the clinical use of opioid analgesics. At a cellular level, MOP regulation occurs via receptor phosphorylation, desensitization, plasma membrane redistribution, and internalization. Here, we used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to detect and quantify ligand-dependent changes in the plasma membrane organization of MOP expressed in human embryonic kidney (HEK293) cells. The low internalizing agonist morphine and the antagonist naloxone did not alter constitutive MOP plasma membrane organization. In contrast, the internalizing agonist DAMGO changed MOP plasma membrane organization in a pertussis toxin-insensitive manner and by two mechanisms. Firstly, it slowed MOP diffusion in a manner that was independent of internalization but dependent on GRK2/3. Secondly, DAMGO reduced the surface receptor number and the proportion of mobile receptors, and increased receptor clustering in a manner that was dependent on clathrin-mediated endocytosis. Overall, these results suggest the existence of distinct sequential MOP reorganization events at the plasma membrane and provide insights into the specific protein interactions that control MOP plasma membrane organization.

Altered membrane rigidity via enhanced endogenous cholesterol synthesis drives cancer cell resistance to destruxins.

Destruxins, secondary metabolites of entomopathogenic fungi, exert a wide variety of interesting characteristics ranging from antiviral to anticancer effects. Although their mode of action was evaluated previously, the molecular mechanisms of resistance development are unknown. Hence, we have established destruxin-resistant sublines of HCT116 colon carcinoma cells by selection with the most prevalent derivatives, destruxin (dtx)A, dtxB and dtxE. Various cell biological and molecular techniques were applied to elucidate the regulatory mechanisms underlying these acquired and highly stable destruxin resistance phenotypes. Interestingly, well-known chemoresistance-mediating ABC efflux transporters were not the major players. Instead, in dtxA- and dtxB-resistant cells a hyper-activated mevalonate pathway was uncovered resulting in increased de-novo cholesterol synthesis rates and elevated levels of lanosterol, cholesterol as well as several oxysterol metabolites. Accordingly, inhibition of the mevalonate pathway at two different steps, using either statins or zoledronic acid, significantly reduced acquired but also intrinsic destruxin resistance. Vice versa, cholesterol supplementation protected destruxin-sensitive cells against their cytotoxic activity. Additionally, an increased cell membrane adhesiveness of dtxA-resistant as compared to parental cells was detected by atomic force microscopy. This was paralleled by a dramatically reduced ionophoric capacity of dtxA in resistant cells when cultured in absence but not in presence of statins. Summarizing, our results suggest a reduced ionophoric activity of destruxins due to cholesterol-mediated plasma membrane re-organization as molecular mechanism underlying acquired destruxin resistance in human colon cancer cells. Whether this mechanism might be valid also in other cell types and organisms exposed to destruxins e.g. as bio-insecticides needs to be evaluated.

Plasma membrane reorganization links acid sphingomyelinase/ceramide to p38 MAPK pathways in endothelial cells apoptosis

The p38 MAPK signaling pathway is essential in the cellular response to stress stimuli, in particular in the endothelial cells that are major target of external stress. The importance of the bioactive sphingolipid ceramide generated by acid sphingomyelinase is also firmly established in stress-induced endothelial apoptotic cell death. Despite a suggested link between the p38 MAPK and ceramide pathways, the exact molecular events of this connection remain elusive. In the present study, by using two different activators of p38 MAPK, namely anisomycin and ionizing radiation, we depicted how ceramide generated by acid sphingomyelinase was involved in p38 MAPK-dependent apoptosis of endothelial cells. We first proved that both anisomycin and ionizing radiation conducted to apoptosis through activation of p38 MAPK in human microvascular endothelial cells HMEC-1. We then found that both treatments induced activation of acid sphingomyelinase and the generation of ceramide. This step was required for p38 MAPK activation and apoptosis. We finally showed that irradiation, as well as treatment with exogenous C16-ceramide or bacterial sphingomyelinase, induced in endothelial cells a deep reorganization of the plasma membrane with formation of large lipid platforms at the cell surface, leading to p38 MAPK activation and apoptosis in endothelial cells. Altogether, our results proved that the plasma membrane reorganization leading to ceramide production is essential for stress-induced activation of p38 MAPK and apoptosis in endothelial cells and established the link between the acid sphingomyelinase/ceramide and p38 MAPK pathways.

Assembly of ER-PM Junctions: A Critical Determinant in the Regulation of SOCE and TRPC1.

Store-operated calcium entry (SOCE), a unique plasma membrane Ca2+ entry mechanism, is activated when ER-[Ca2+] is decreased. SOCE is mediated via the primary channel, Orai1, as well as others such as TRPC1. STIM1 and STIM2 are ER-Ca2+ sensor proteins that regulate Orai1 and TRPC1. SOCE requires assembly of STIM proteins with the plasma membrane channels which occurs within distinct regions in the cell that have been termed as endoplasmic reticulum (ER)-plasma membrane (PM) junctions. The PM and ER are in close proximity to each other within this region, which allows STIM1 in the ER to interact with and activate either Orai1 or TRPC1 in the plasma membrane. Activation and regulation of SOCE involves dynamic assembly of various components that are involved in mediating Ca2+ entry as well as those that determine the formation and stabilization of the junctions. These components include proteins in the cytosol, ER and PM, as well as lipids in the PM. Recent studies have also suggested that SOCE and its components are compartmentalized within ER-PM junctions and that this process might require remodeling of the plasma membrane lipids and reorganization of structural and scaffolding proteins. Such compartmentalization leads to the generation of spatially- and temporally-controlled Ca2+signals that are critical for regulating many downstream cellular functions.

Structural and Dynamic Study of Caveolin-1 Membrane Nanodomains in Response to Inflammatory Pathway Signaling

Within the plasma membrane, the protein caveolin-1 (cav-1) forms various diffraction-limited microdomains that have yet to been precisely characterized in terms of structure and dynamics. Cav-1 is an important regulator of the GPCR protease activated receptor-1 (PAR-1), notably during the activation of cellular inflammation responses. The regulatory role of cav-1 during inflammatory responses is linked to the differential activation of RhoA and Rac1 signaling pathways and to cytoskeletal rearrangements that result in wide scale plasma membrane reorganization and cell migration. Using single molecule and super-resolution imaging techniques coupled with quantitative spatial clustering analyses, we study the membrane distribution and the dynamics of caveolae and flat cav-1 scaffolds in response to PAR-1 pathway activations. Cav-1 knock-out MEF and iMAEC cells were rescued with cav-1 fusions to SNAP tag and immunolabeled against endogenous PAR-1 for dual color 3D super-resolution imaging of individual cav-1 and PAR-1 proteins. PAR-1 was directly stimulated using site-specific activating peptides to trigger inflammatory response cascades. At the plasma membrane, 3D super-resolution imaging revealed the expected ∼100 nm 3D globular shape of caveolae as well as flat cav-1 rich nanodomains with structures matching the expected size and z-dimensional range of cav-1 scaffolds. Using robust auto- and cross-correlation statistical analyses of individual cav-1 and PAR-1 proteins spatially mapped with a precision <10 nm, we show that the distribution, the number and the size of cav-1 nanodomains change dynamically in response to either pro- or anti- inflammatory signaling as does the interconnectivity between caveolae, cav-1 scaffolds and PAR-1 receptors at the cell surface. We are able to couple the molecular re-organization of diffraction-limited cav-1 nanodomains with broader cytoskeletal and plasma membrane remodeling in response to defined signaling cues during cellular wound healing.

ADP-Ribosylation Factor 1 Regulates Proliferation, Migration, and Fusion in Early Stage of Osteoclast Differentiation.

Small G-protein adenosine diphosphate (ADP)-ribosylation factors (ARFs) regulate a variety of cellular functions, including actin cytoskeleton remodeling, plasma membrane reorganization, and vesicular transport. Here, we propose the functional roles of ARF1 in multiple stages of osteoclast differentiation. ARF1 was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and transiently activated in an initial stage of their differentiation. Differentiation of ARF1-deficient osteoclast precursors into mature osteoclasts temporarily increased in pre-maturation stage of osteoclasts followed by reduced formation of mature osteoclasts, indicating that ARF1 regulates the osteoclastogenic process. ARF1 deficiency resulted in reduced osteoclast precursor proliferation and migration as well as increasing cell-cell fusion. In addition, ARF1 silencing downregulated c-Jun N-terminal kinase (JNK), Akt, osteopontin, and macrophage colony-stimulating factor (M-CSF)-receptor c-Fms as well as upregulating several fusion-related genes including CD44, CD47, E-cadherin, and meltrin-α. Collectively, we showed that ARF1 stimulated proliferation and migration of osteoclast precursors while suppressing their fusion, suggesting that ARF1 may be a plausible inter-player that mediates the transition to osteoclast fusion at multiple steps during osteoclast differentiation

Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements

Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by ‘Candidatus Phytoplasma solani,’ the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Western blot analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes.

Blocking integrin inactivation as an anti-angiogenic therapy.

During angiogenesis, endothelial cell migration is coordinated by integrin‐mediated contact with the extra‐cellular matrix (ECM), coupled with receptor tyrosine kinase signalling to regulate dynamic cytoskeletal and plasma membrane reorganization. A recent paper by Vitorino et al (2015) defined a new MAP4K4–moesin–talin–β1‐integrin pathway that could be therapeutically exploited to suppress pathologic angiogenesis.

Visualizing Actin Architectures in Cells Incubated with Cell-Penetrating Peptides.

Defining the exact role of the actin cytoskeleton in mediating endocytosis through different pathways is a significant challenge. The general consensus is that actin has an important role in organizing the early stages of endocytosis but there is still much to learn. Actin has also been implicated in cell internalization of cell-penetrating peptides (CPPs). It is suggested that CPP variants such as octaarginine (R8) and the HIV Tat peptide induce actin-dependent plasma membrane perturbation and enter via macropinocytosis. Here, we describe confocal microscopy techniques that allow for high-resolution spatial characterization of the actin cytoskeleton in untreated mammalian cells and those incubated with actin-disrupting agents and CPPs. By performing X-Y-Z projection images through different regions of cells to show basal and apical profiles, we initially highlight how these techniques can be used to reveal major differences in cortical and filamentous actin organization between different cell lines. Using these techniques, we demonstrate that the actin-disrupting agent cytochalasin D rapidly changes this framework at concentrations significantly lower than is normally used. Experiments are also performed to highlight that serum starvation significantly sensitizes cells to the effects of R8 on actin-induced ruffling and lamellapodia formation. The techniques described here can be used to gain a higher level of knowledge of the organization of the actin network in individual model cell systems, how this is perturbed using commonly used actin inhibitors, and how plasma membrane reorganization can be induced by the addition of drug delivery vectors such as CPPs.

Plasma membrane reorganization: A glycolipid gateway for microbes

Ligand–receptor interactions, which represent the core for cell signaling and internalization processes are largely affected by the spatial configuration of host cell receptors. There is a growing piece of evidence that receptors are not homogeneously distributed within the plasma membrane, but are rather pre-clustered in nanodomains, or clusters are formed upon ligand binding. Pathogens have evolved many strategies to evade the host immune system and to ensure their survival by hijacking plasma membrane receptors that are most often associated with lipid rafts. In this review, we discuss the early stage molecular and physiological events that occur following ligand binding to host cell glycolipids. The ability of various biological ligands (e.g. toxins, lectins, viruses or bacteria) that bind to glycolipids to induce their own uptake into mammalian cells by creating negative membrane curvature and membrane invaginations is explored. We highlight recent trends in understanding nanoscale plasma membrane (re-)organization and present the benefits of using synthetic membrane systems. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

The number of Neisseria meningitidis type IV pili determines host cell interaction.

As mediators of adhesion, autoaggregation and bacteria-induced plasma membrane reorganization, type IV pili are at the heart of Neisseria meningitidis infection. Previous studies have proposed that two minor pilins, PilV and PilX, are displayed along the pilus structure and play a direct role in mediating these effects. In contrast with this hypothesis, combining imaging and biochemical approaches we found that PilV and PilX are located in the bacterial periplasm rather than along pilus fibers. Furthermore, preventing exit of these proteins from the periplasm by fusing them to the mCherry protein did not alter their function. Deletion of the pilV and pilX genes led to a decrease in the number, but not length, of pili displayed on the bacterial surface indicating a role in the initiation of pilus biogenesis. By finely regulating the expression of a central component of the piliation machinery, we show that the modest reductions in the number of pili are sufficient to recapitulate the phenotypes of the pilV and pilX mutants. We further show that specific type IV pili-dependent functions require different ranges of pili numbers.

Early sequence of events triggered by the interaction ofNeisseria meningitidiswith endothelial cells

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus-mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high-resolution quantitative live-cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili-associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl-β-cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria-induced plasma membrane reorganization is a rapid event driven by a direct cross-talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.

Phosphoinositide 3-kinase at the crossroad between endocytosis and signaling of cytokine receptors

Class I phosphoinositide 3-kinase (PI3K) is a lipid kinase playing key roles in many signaling pathways regulating cell survival and growth. Besides its important role in signal transduction, PI3K is also involved in actin and membrane reorganization such as protrusion, adhesion, phagocytosis and macropinocytosis. Receptor-mediated endocytosis is initiated by plasma membrane reorganization creating buds that then mature to small vesicles. Whereas most of endocytic mechanisms involve actin polymerization, PI3K requirement has not been clearly investigated. Our study identifies class I PI3K as a key player in clathrin-independent endocytosis of the interleukin 2 receptor (IL-2R) in contrast to the clathrin-dependent entry of transferrin (Tf). IL-2R is a cytokine receptor, inducing several signaling cascades such as PI3K, that are essential for the immune response. We have shown previously that IL-2R can be internalized with or without IL-2 and this process requires dynamin, actin and their regulators cortactin, N-WASP, Rac1 and the kinases Pak. Our recent work reveals that PI3K regulates Rac1 during IL-2R uptake in two ways: via its catalytic activity (p110) and via its regulatory factor (p85). Indeed, the catalytic activity of PI3K is required for both constitutive and IL-2 induced uptake of cytokine receptors, in lymphocytes as well as in epithelial cells. Interestingly, Vav2, a Rac1 GTPase exchange factor (GEF) induced upon PI3K activation, is specifically involved and recruited during IL-2R uptake. The second action of PI3K is via its regulatory subunit, p85, which binds activated Rac1 and IL-2R; this interaction being enhanced upon IL-2 treatment. Thus, PI3K regulates both the activation of Rac1 and its recruitment during IL-2R endocytosis. Finally, our results identify a link between cytokine receptors signaling and clathrin-independent endocytosis.

ADP-Ribosylated Proteins as Old and New Drug Targets for Anticancer Therapy: The Example of ARF6

Post-translational modifications of cellular proteins by mono- or poly-ADP-ribosylation are associated with numerous cellular processes. ADP-ribosylation reactions are important in the nucleus, and in mitochondrial activity, stress response signaling, intracellular trafficking, and cell senescence and apoptosis decisions. These reversible reactions add ADP-ribose to target proteins via specific enzymes to form the ADP-ribosylated protein; the cleaveage of this covalent bond is performed via hydrolases. Deficiencies in these enzymatic activities lead to cell death or tumor formation, thus defining their functional roles and impact on human disease. Unlike mono- ADP-ribosyltransferases, poly-ADP-ribose polymerases (PARPs) have been at the frontline of drug discovery since the 1980s. PARP1 is a valuable therapeutic target, with a central role in responses to DNA damage. With mono-ADP-ribosylation now linked to human diseases, such as inflammation, diabetes, neurodegeneration and cancer metastasis, novel and equally important functions of mono-ADPribosylation in cell signaling pathways can now be defined. Recently, we reported mono-ADP-ribosylation of ADP-ribosylation factor 6 (ARF6), a small G-protein of the Ras superfamily. In addition to its involvement in actin remodeling, plasma membrane reorganization and vesicular transport, ARF6 contributes to cancer progression through activation of cell motility and invasion. Consequently, targeting this modification will counteract the pro-invasive effects of ARF6, providing innovative anti-tumor therapy. This review summarizes our present knowledge of the enzymes and targets involved in ADP-ribosylation reactions, and describes in silico approaches to visualize their site of interaction and to identify the precise site for ADP-ribosylation. This should ultimately improve pharmacological strategies to enhance both anti-tumor efficacy and treatment of a number of inflammatory and neurodegenerative disorders.