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•DDR and repair mechanisms have evolved to prevent harmful effects of DNA damage.•Typical Rho GTPases regulate cytoplasmic and nuclear actin dynamics.•Rho, Rac and Cdc42 are important ...and quasi-direct regulators of genomic stability.•DNA damage is a hallmark of cellular disorders such as cancer and premature aging.•Rho GTPases signaling are new therapeutic windows for pathophysiological disorders.
The classical small Rho GTPase (Rho, Rac, and Cdc42) protein family is mainly responsible for regulating cell motility and polarity, membrane trafficking, cell cycle control, and gene transcription. Cumulative recent evidence supports important roles for these proteins in the maintenance of genomic stability. Indeed, DNA damage response (DDR) and repair mechanisms are some of the prime biological processes that underlie several disease phenotypes, including genetic disorders, cancer, senescence, and premature aging. Many reports guided by different experimental approaches and molecular hypotheses have demonstrated that, to some extent, direct modulation of Rho GTPase activity, their downstream effectors, or actin cytoskeleton regulation contribute to these cellular events. Although much attention has been paid to this family in the context of canonical actin cytoskeleton remodeling, here we provide a contextualized review of the interplay between Rho GTPase signaling pathways and the DDR and DNA repair signaling components. Interesting questions yet to be addressed relate to the spatiotemporal dynamics of this collective response and whether it correlates with different subcellular pools of Rho GTPases. We highlight the direct and indirect targets, some of which still lack experimental validation data, likely associated with Rho GTPase activation that provides compelling evidence for further investigation in DNA damage-associated events and with potential therapeutic applications in translational medicine.
Vaccinia virus is a poxvirus that has been successfully leveraged to develop vaccines for smallpox, which is caused by the closely related Variola virus. Smallpox has been declared as 'eradicated' by ...the WHO in 1980; however, it still poses a potential bioterrorism threat. More recently, the spreading of monkeypox (MPox) in non-endemic countries has further highlighted the importance of continuing the exploration for druggable targets for poxvirus infections. The vaccinia H1 (VH1) phosphatase is the first reported dual specificity phosphatase (DUSP) able to hydrolyze both phosphotyrosine and phosphoserine/phosphotheonine residues. VH1 is a 20 kDa protein that forms a stable dimer and can dephosphorylate both viral and cellular substrates to regulate the viral replication cycle and host immune response. VH1 dimers adopt a domain swap mechanism with the first 20 amino acids of each monomer involved in dense electrostatic interaction and salt bridge formations while hydrophobic interactions between the N-terminal and C-terminal helices further stabilize the dimer. VH1 appears to be an ideal candidate for discovery of novel anti-poxvirus agents because it is highly conserved within the poxviridae family and is a virulence factor, yet it displays significant divergence in sequence and dimerization mechanism from its human closest ortholog vaccinia H1-related (VHR) phosphatase, encoded by the DUSP3 gene. As the dimeric quaternary structure of VH1 is essential for its phosphatase activity, strategies leading to disruption of the dimer structure might aid in VH1 inhibitor development.
Intracellular peptides are produced by proteasomes following degradation of nuclear, cytosolic, and mitochondrial proteins, and can be further processed by additional peptidases generating a larger ...pool of peptides within cells. Thousands of intracellular peptides have been sequenced in plants, yeast, zebrafish, rodents, and in human cells and tissues. Relative levels of intracellular peptides undergo changes in human diseases and also when cells are stimulated, corroborating their biological function. However, only a few intracellular peptides have been pharmacologically characterized and their biological significance and mechanism of action remains elusive. Here, some historical and general aspects on intracellular peptides' biology and pharmacology are presented. Hemopressin and Pep19 are examples of intracellular peptides pharmacologically characterized as inverse agonists to cannabinoid type 1 G-protein coupled receptors (CB1R), and hemopressin fragment NFKF is shown herein to attenuate the symptoms of pilocarpine-induced epileptic seizures. Intracellular peptides EL28 (derived from proteasome 26S protease regulatory subunit 4; Rpt2), PepH (derived from Histone H2B type 1-H), and Pep5 (derived from G1/S-specific cyclin D2) are examples of peptides that function intracellularly. Intracellular peptides are suggested as biological functional molecules, and are also promising prototypes for new drug development.
Actin cytoskeleton remodeling is the major motor of cytoskeleton dynamics driving tumor cell adhesion, migration and invasion. The typical RhoA, RhoB and RhoC GTPases are the main regulators of actin ...cytoskeleton dynamics. The C3 exoenzyme transferase from Clostridium botulinum is a toxin that causes the specific ADP-ribosylation of Rho-like proteins, leading to its inactivation. Here, we examine what effects the Rho GTPase inhibition and the consequent actin cytoskeleton instability would have on the emergence of DNA damage and on the recovery of genomic stability of malignant melanoma cells, as well as on their survival. Therefore, the MeWo cell line, here assumed as a melanoma cell line model for the expression of genes involved in the regulation of the actin cytoskeleton, was transiently transfected with the C3 toxin and subsequently exposed to UV-radiation. Phalloidin staining of the stress fibers revealed that actin cytoskeleton integrity was strongly disrupted by the C3 toxin in association with reduced melanoma cells survival, and further enhanced the deleterious effects of UV light. MeWo cells with actin cytoskeleton previously perturbed by the C3 toxin still showed higher levels and accumulation of UV-damaged DNA (strand breaks and cyclobutane pyrimidine dimers, CPDs). The interplay between reduced cell survival and impaired DNA repair upon actin cytoskeleton disruption can be explained by constitutive ERK1/2 activation and an inefficient phosphorylation of DDR proteins (γH2AX, CHK1 and p53) caused by C3 toxin treatment. Altogether, these results support the general idea that actin network help to protect the genome of human cells from damage caused by UV light through unknown molecular mechanisms that tie the cytoskeleton to processes of genomic stability maintenance.
•Specific targeting of Rho GTPases by C3 toxin disrupts actin cytoskeleton.•Genotoxic stress promoted by UV light affects the integrity of actin cytoskeleton.•C3 toxin hypersensitizes melanoma cells to the UV-radiation effects reducing survival.•The actin cytoskeleton integrity is directly linked to genomic stability.•C3 toxin associated with UV stress reduces DDR signaling an increases MAPK signaling.
Background Information
The dual‐specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase ...interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC‐ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme.
Results
The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans‐acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr‐phosphorylated HNRNPC preferentially binds to IRES‐containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c‐MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated‐HNRNPC/RNA interaction reduces HNRNPC‐p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA‐dependent manner.
Conclusions and Significance
Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC‐ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM‐domain proteins.
DUSP3 knockdown increases tyrosine phosphorylation levels of HNRNPC. Phosphorylated HNRNPC has increased capacity for RNA recognition and binding but reduced p53 binding. DUSP3 is present in the 40S subunit of ribosome, monosomes and polysomes interacting with HNRNPC. In DUSP3‐depleted cells the phosphorylated HNRNPC preferentially binds IRES containing mRNAs such as c‐myc and xiap increasing their protein levels, but not their mRNAs.
Dual Specificity Phosphatase 12 is a member of the Atypical DUSP Protein Tyrosine Phosphatase family, meaning that it does not contain typical MAP kinase targeting motifs, while being able to ...dephosphorylate tyrosine and serine/threonine residues. DUSP12 contains, apart from its catalytic domain, a zinc finger domain, making it one of the largest DUSPs, which displays strong nuclear expression in several tissues. In this work we identified nuclear targets of DUSP12 in two different cancer cell lines (A549 and MCF-7), challenging them with genotoxic stimuli to observe the effect on the networks and to link existing information about DUSP12 functions to the data obtained though mass spectrometry. We found network connections to the cytoskeleton (e.g. IQGAP1), to the chromatin (e.g. HP1BP3), to the splicing machinery and to the previously known pathway of ribosome maturation (e.g. TCOF1), which draw insight into many of the functions of this phosphatase, much likely connecting it to distinct, previously unknown genomic stability mechanisms.
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•DUSP12 partners in the nucleus are affected by genotoxic stimuli.•Network analyses point to interactions with ribosomal maturation and stress granules.•Newfound connections to cytoskeleton, chromatin and RNA processing machinery.•Co-precipitation indicates interaction with TCOF1-containing particles and NAT10.•Confocal fluorescence shows colocalization with HP1BP3-enriched chromatin regions.
Cdc42, a member of the Rho GTPase family, is an intracellular signaling protein known for its roles in cytoskeleton rearrangements and, more recently, in apoptosis/senescence triggered by genotoxic ...stress. In some tumor cells, the overactivation of Cdc42 through the expression of constitutively active mutants (G12V or Q61L), GEF activation, or GAP downregulation functions as an antiproliferative or pro-aging mechanism. In this study, human cell lines with different P53 protein profiles were exposed to UV radiation, and the interactions between Cdc42 and proteins that are putatively involved in the DNA damage response and repair mechanisms were screened. The affinity-purified proteins obtained through pull-down experiments of the cell lysates using the recombinant protein baits GST, GST-Cdc42-WT, or GST-Cdc42-G12V were identified by mass spectrometry. The resulting data were filtered and used for the construction of protein–protein interaction networks. Among several promising proteins, three targets, namely, PAK4, PHB-2, and 14-3-3η, which are involved in the cell cycle, apoptosis, DNA repair, and chromatin remodeling processes, were identified. Biochemical validation experiments showed physical and proximal interactions between Cdc42 and the three targets in the cells, particularly after exposure to UV. The results suggest that the molecular mechanisms coordinated by overactivated Cdc42 (with the G12V mutation) to increase the cellular sensitivity to UV radiation and the susceptibility to cell death are collectively mediated by these three proteins. Therefore, the Cdc42 GTPase can potentially be considered another player involved in maintenance of the genomic stability of human cells during exposure to genotoxic stress.
The atypical dual-specificity phosphatases (aDUSPs) are a group of protein tyrosine phosphatases (PTPs) that have been increasingly studied recently, but little is known about their substrates or ...their roles and regulation. aDUSPs are typically low-molecular-weight enzymes that are distinct from the mitogen-activated protein kinase phosphatases (MKPs) but that still function in the regulation of the MAPK signalling cascade. aDUSPs may also have non-MAPK substrates, based on homologies observed in the sequences flanking potential phosphotyrosine target sites of other proteins and the cell type-specific characteristics of certain aDUSPs. Here, we combined experimental and computational tools to identify new substrates and protein partners of VHR (DUSP3) phosphatase in HeLa cells exposed to genotoxic stress. Experimental approaches confirmed the good stability of VHR and its nuclear co-localisation with classical MAPK substrates. The bioinformatics analysis of 4539 human nuclear proteins to identify a subset with functions related to DNA damage response and repair or to checkpoints and cell cycle control, that contain the phosphorylatable Thr-X-Tyr motif of MAPK with a high probability of dual phosphorylation, and that have structural homology to the MAPK activation loop resulted in a list of 57 putative VHR substrates. Fluorescence confocal microscopy and pull-down experiments followed by immunoblots revealed that VHR co-localised and interacted with components of the MRN complex and pH2AX, a DNA double-strand break sensor. Our platform, which combines experimental data from structure-function and bioinformatics analyses based on MAPK substrate similarities, provides a low-cost and rapid approach for the identification of novel aDUSP-interacting proteins with unknown roles in genotoxic stress response and genome stability maintenance.
A comparison among lowering rates collected in the limestones of the northeastern Adriatic Region (Italy and Croatia) gives a picture of the erosion patterns in the inland and coastal Classical Karst ...and Istrian Karst. Erosion rates were measured using either a micro-erosion meter or a traversing micro-erosion meter. Beginning from a large dataset spread over a maximum of 26 years, significant differences in limestone lowering rates have been recognized in the surveyed area: the mean lowering rate for the inland Classical Karst area is 0.018 mm/yr whereas in the inland Istrian Karst it is 0.009 mm/yr. In the coastal sectors, the mean erosion rate varies between 0.14 mm/yr in the Gulf of Trieste and 0.04 mm/yr in the sites located along the Istrian shoreline. These differences are probably due to the climatic setting of the Classical Karst area (Mediterranean Continental to Sub-alpine climate) and Istrian Peninsula (Mediterranean climate). Moreover, along the coasts of the Gulf of Trieste there are many submarine springs which are absent along the Istrian coast. It is hypothesized that these underwater springs stress the effect of rainfall along the coast, which is both direct (rain on the measurement site) and mainly indirect (freshwater outflow from submarine springs). Higher lowering rates were surveyed during autumn; rainfall, therefore also freshwater outflow from submarine springs, is particularly high at the end of the summer and in autumn.
Radiotherapy causes the regression of many human tumors by increasing DNA damage, and the novel molecular mechanisms underlying the genomic instability leading to cancer progression and metastasis ...must be elucidated. Atypical dual-specificity phosphatase 3 (DUSP3) has been shown to down-regulate mitogen-activated protein kinases (MAPKs) to control the proliferation and apoptosis of human cancer cells. We have recently identified novel molecular targets of DUSP3 that function in DNA damage response and repair; however, whether DUSP3 affects these processes remains unknown.
Tumor cell lines in which DUSP3 activity was suppressed by pharmacological inhibitors or a targeted siRNA were exposed to gamma radiation, and proliferation, survival, DNA strand breaks and recombination repair pathways were sequentially analyzed.
The combination of reduced DUSP3 activity and gamma irradiation resulted in decreased cellular proliferation and survival and increased cellular senescence compared with the effects of radiation exposure alone. Gamma radiation-induced DNA damage was increased by the loss of DUSP3 activity and correlated with increased levels of phospho-H2AX protein and numbers of ionizing radiation-induced γ-H2AX foci, which were reflected in diminished efficiencies of homologous recombination (HR) and non-homologous end-joining (NHEJ) repair. Similar results were obtained in ATM-deficient cells, in which reduced DUSP3 activity increased radiosensitivity, independent of increased MAPK phosphorylation.
The loss of DUSP3 activity markedly increases gamma radiation-induced DNA strand breaks, suggesting a potential novel role for DUSP3 in DNA repair.
The radioresistance of tumor cells is effectively reduced by a combination of approaches through the inhibition of DUSPs.
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•DUSP3 inhibition or knockdown increases DNA damage caused by gamma radiation.•DUSP3 inhibition or knockdown reduces DNA repair in tumor cells.•DUSP3 inhibition or knockdown reduces the proliferation and survival of tumor cells.•DUSP3 inhibition increases the cellular senescence of tumor cells.•The effects of the loss of DUSP3 activity are more apparent in ATM-deficient cells.
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