Targeting Rap1b signaling cascades with CDNF: Mitigating platelet activation, plasma oxylipins and reperfusion injury in stroke.

Cerebral reperfusion injury in stroke, stemming from interconnected thrombotic and inflammatory signatures, often involves platelet activation, aggregation and its interaction with various immune cells, contributing to microvascular dysfunction. However, the regulatory mechanisms behind this platelet activation and the resulting inflammation are not well understood, complicating the development of effective stroke therapies. Utilizing animal models and platelets from hemorrhagic stroke patients, our research demonstrates that human cerebral dopamine neurotrophic factor (CDNF) acts as an endogenous antagonist, mitigating platelet aggregation and associated neuroinflammation. CDNF moderates mitochondrial membrane potential, reactive oxygen species production, and intracellular calcium in activated platelets by interfering with GTP binding to Rap1b, thereby reducing Rap1b activation and downregulating the Rap1b-MAPK-PLA2 signaling pathway, which decreases release of the pro-inflammatory mediator thromboxane A2. In addition, CDNF reduces the inflammatory response in BV2 microglial cells co-cultured with activated platelets. Consistent with ex vivo findings, subcutaneous administration of CDNF in a rat model of ischemic stroke significantly reduces platelet activation, aggregation, lipid mediator production, infarct volume, and neurological deficits. In summary, our study highlights CDNF as a promising therapeutic target for mitigating platelet-induced inflammation and enhancing recovery in stroke. Harnessing the CDNF pathway may offer a novel therapeutic strategy for stroke intervention.

Novel Pt(IV) complexes containing salvigenin ligand reverse cisplatin-induced resistance by inhibiting Rap1b-mediated cancer cell stemness in esophageal squamous cell carcinoma treatments.

Esophageal squamous cell carcinoma (ESCC) is a malignant tumor that is highly susceptible to metastasis, recurrence and resistance, and few therapeutic targets have been identified and proven effective. Herein, we demonstrated for the first time that Rap1b can positively regulate ESCC cell stemness, as well as designed and synthesized a novel class of Pt(IV) complexes that can effectively inhibit Raplb. In vitro biological studies showed that complex-1 exhibited stronger cytotoxicity than cisplatin and oxaliplatin against a variety of ESCC cells, and effectively reversed cisplatin-induced resistance of TE6 cells by increasing cellular accumulation of platinum and inhibiting cancer cell stemness. Significantly, complex-1 also exhibited strong ability to reversal cisplatin-induced cancer cell resistance and inhibit tumor growth in TE6/cDDP xenograft mice models, with a tumor growth inhibition rate of 73.3 % at 13 mg/kg and did not show significant systemic toxicity. Overall, Rap1b is a promising target to be developed as an effective treatment for ESCC. Complex-1, as the first Pt(IV) complex that can strongly inhibit Rap1b, is also worthy of further in-depth study.

MicroR-380-3p Reduces Sepsis-Induced Acute Kidney Injury via Regulating RAB1P to Restrain NF-κB Pathway.

Septic acute kidney injury (AKI) is a common complication in critically ill patients with high morbidity and mortality. This study intends to clarify the clinical value and molecular mechanism of microR-380-3p in septic AKI by recruiting patients with septic AKI and establishing septic AKI cell models. Patients with septic AKI were included and human kidney-2 (HK-2) cells were induced by lipopolysaccharide (LPS) to construct the AKI cell model of sepsis. The expression of microR-380-3p was detected by quantitative real-time RT-PCR (qRT-PCR). The expression of Bax, cleaved caspase 3, Bcl-2, p65, and p-p65 was detected by Western blot. The contents of inflammation and oxidation were determined by commercial kits. Bioinformatics predicted the binding target of microR-380-3p and a dual luciferase reporting system was used to verify the regulatory relationship between microR-380-3p and RAP1B. The concentration of microR-380-3p was elevated in patients with septic AKI and appeared to be a biomarker for these patients. Silenced microR-380-3p reversed the damage of LPS on HK-2 cells via promoting viability, inhibiting apoptosis, inflammation, and oxidation. RAP1B was a target of microR-380-3p and microR-380-3p exerted targeted inhibition of RAP1B expression level. Down-regulation of RAP1B reversed the influence of silenced microR-380-3p on HK-2 cells. MicroR-380-3p/RAP1B participated in activating the NF-κB pathway. MicroR-380-3p down-regulated RAP1B to exacerbate septic AKI, providing a potential therapeutic biomarker for septic AKI.

Adding to the evidence of gene-disease association of RAP1B and syndromic thrombocytopenia.

Syndromic constitutive thrombocytopenia encompasses a heterogeneous group of disorders characterised by quantitative and qualitative defects of platelets while featuring other malformations. Recently, heterozygous, de novo variants in RAP1B were reported in three cases of syndromic thrombocytopenia. Here, we report two additional, unrelated individuals identified retrospectively in our data repository with heterozygous variants in RAP1B: NM_001010942.2(RAP1B):c.35G>A, p.(Gly12Glu) (de novo) and NM_001010942.2(RAP1B):c.178G>A, p.(Gly60Arg). Both individuals had thrombocytopenia, as well as congenital malformations, and neurological, behavioural, and dysmorphic features, in line with previous reports. Our data supports the causal role of monoallelic RAP1B variants that disrupt RAP1B GTPase activity in syndromic congenital thrombocytopenia.

Host cell cAMP-Epac-Rap1b pathway inhibition by hawthorn extract as a potential target against Trypanosoma cruzi infection.

Although the two drugs currently available for the treatment of Chagas disease, Benznidazole and Nifurtimox, have proven to be effective in the acute phase of the disease, the 60-90-day treatment leads to high toxicity and unwanted side effects, presenting, in addition, a low efficacy in the chronic phase of the disease. For this reason, new therapies that are more effective are needed. In this regard, we have recently shown that the inhibition of the Epac-Rap1b pathway suppressed the cAMP-mediated host cell invasion by Trypanosoma cruzi. Interestingly, it has been described that vitexin, a natural flavone that protects against ischemia-reperfusion damage, acts by inhibiting the expression of Epac and Rap1 proteins. Vitexin can be found in plants of the genus Crataegus spp., traditionally known as hawthorn, which are of great interest considering their highly documented use as cardio-protectors. Pre-treating cells with an extract of Crataegus oxyacantha produced levels of T. cruzi invasion comparable to the ones observed for the commercially available Epac1-specific inhibitor, ESI-09. In addition, extract-treated cells exhibited a decrease in the activation of Rap1b, suggesting that the effects of the extract would be mediated by the inhibition of the cAMP-Epac-Rap1 signaling pathway. Using HPLC-HRMS2, we could confirm the presence of vitexin, and other flavones that could act as inhibitors of Epac/Rap1b, in the extracts of C. oxyacantha. Most significantly, when cells were treated with the extract of C. oxyacantha in conjunction with Nifurtimox, an increased modulation of invasion was observed.

[Molecular dynamics simulation of force-regulated interaction between talin and Rap1b].

The binding of talin-F0 domain to ras-related protein 1b (Rap1b) plays an important role in the formation of thrombosis. However, since talin is a force-sensitive protein, it remains unclear whether and how force regulates the talin-F0/Rap1b interaction. To explore the effect of force on the binding affinity and the dynamics mechanisms of talin-F0/Rap1b, molecular dynamics simulation was used to observe and compare the changes in functional and conformational information of the complex under different forces. Our results showed that when the complex was subjected to tensile forces, there were at least two dissociation pathways with significantly different mechanical strengths. The key event determining the mechanical strength difference between the two pathways was whether the ß4 sheet of the F0 domain was pulled away from the original ß1-ß4 parallel structure. As the force increased, the talin-F0/Rap1b interaction first strengthened and then weakened, exhibiting the signature of a transition from catch bonds to slip bonds. The mechanical load of 20 pN increased the interaction index of two residue pairs, ASP 54-ARG 41 and GLN 18-THR 65, which resulted in a significant increase in the affinity of the complex. This study predicts the regulatory mechanism of the talin-F0/Rap1b interaction by forces in the intracellular environment and provides novel ideas for the treatment of related diseases and drug development.

Plasma biomarker panel for major depressive disorder by quantitative proteomics using ensemble learning algorithm: A preliminary study.

Major depressive disorder (MDD) is a major international public health issue; thus, investigating its underlying mechanisms and identifying suitable biomarkers to enable its early detection are imperative. Using data-independent acquisition-mass spectrometry-based proteomics, the plasma of 44 patients with MDD and 25 healthy controls was studied to detect differentially expressed proteins. Bioinformatics analyses, such as Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, Protein-Protein Interaction network, and weighted gene co-expression network analysis were employed. Moreover, an ensemble learning technique was used to build a prediction model. A panel of two biomarkers, L-selectin and an isoform of the Ras oncogene family was identified. With an area under the receiver operating characteristic curve of 0.925 and 0.901 for the training and test sets, respectively, the panel was able to distinguish MDD from the controls. Our investigation revealed numerous potential biomarkers and a diagnostic panel based on several algorithms, which may contribute to the future development of a plasma-based diagnostic approach and better understanding of the molecular mechanisms of MDD.

Circ_0000231 promotes paclitaxel resistance in ovarian cancer by regulating miR-140/RAP1B.

Circular RNAs (circRNAs) are identified as vital regulators in a variety of cancers. However, the involvement of circ_0000231 in paclitaxel (PTX) resistant ovarian cancer (OC) remains unclear. In this study, we examined the levels of circ_0000231, microRNA-140 (miR-140) and RAP1B in PTX-resistant OC tissues and cells and found that circ_0000231 and RAP1B levels were increased, while miR-140 level was decreased in these cells. Depletion of circ_0000231 could inhibit the resistance, proliferation, invasion, migration and EMT and promoted the apoptosis of PTX-resistant OC cells. The opposite effects were observed by overexpression of circ_0000231. Furthermore, the effect of circ_0000231 on the PTX sensitivity of OC cells was investigated by using xenograft tumor models, and circ_0000231 knockdown increased PTX sensitivity of OC in vivo. Mechanistically, we demonstrated that circ_0000231 acted as a sponge for miR-140, and RAP1B was the target gene of miR-140. Taken together, these data indicated that circ_0000231 was a key molecule required for the growth, migration, and PTX-resistance of OC cells and was involved in EMT. Knockdown of circ_000231 suppressed PTX-resistant OC progression via regulating miR-140/RAP1B signaling pathway. circ_0000231 might play vital roles in the tumorigenesis and chemoresistance of OC.

Roles of the hsa_circ_0013880/USP32/Rap1b axis in the proliferation and apoptosis of acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a myeloid malignancy with generally high mortality. Although recent advances in AML research have revealed that circRNAs play significant roles in AML progression, our understanding of the leukemogenic mechanism of circRNAs remains very limited. In this study, increased expression of hsa_circ_0013880 was observed in bone marrow mononuclear cells (BMNCs) of AML patients. Overexpression of hsa_circ_0013880 promotes AML cell proliferation and migration and reduces cell apoptosis. Mechanistically, hsa_circ_0013880 could elevate the expression of USP32, a deubiquitinating enzyme that is highly expressed in the BMNCs of AML patients. Given the deubiquitination function of USP32, we further hypothesize that USP32 may mediate the malignant behaviors of AML cells by regulating the stability of Ras-related protein (Rap1b). At the molecular level, we find that silencing of USP32 increases ubiquitinated Rap1b. Overexpression of Rap1b restores the effects of USP32 knockdown, which further verifies our hypothesis. In addition, we propose another hypothesis that a potential regulatory network among hsa_circ_0013880, miR-148a-3p/miR-20a-5p and USP32 might exist in the development of AML, according to bioinformatics website predictions and our preliminary experimental verification. Overall, our findings will enrich the understanding of the hsa_circ_0013880/USP32/Rap1b axis in AML development, which may contribute to the development of novel therapeutic strategies for AML.

Molecular dynamics simulation of force-regulated interaction between talin and Rap1b / 生物医学工程学杂志

The binding of talin-F0 domain to ras-related protein 1b (Rap1b) plays an important role in the formation of thrombosis. However, since talin is a force-sensitive protein, it remains unclear whether and how force regulates the talin-F0/Rap1b interaction. To explore the effect of force on the binding affinity and the dynamics mechanisms of talin-F0/Rap1b, molecular dynamics simulation was used to observe and compare the changes in functional and conformational information of the complex under different forces. Our results showed that when the complex was subjected to tensile forces, there were at least two dissociation pathways with significantly different mechanical strengths. The key event determining the mechanical strength difference between the two pathways was whether the β4 sheet of the F0 domain was pulled away from the original β1-β4 parallel structure. As the force increased, the talin-F0/Rap1b interaction first strengthened and then weakened, exhibiting the signature of a transition from catch bonds to slip bonds. The mechanical load of 20 pN increased the interaction index of two residue pairs, ASP 54-ARG 41 and GLN 18-THR 65, which resulted in a significant increase in the affinity of the complex. This study predicts the regulatory mechanism of the talin-F0/Rap1b interaction by forces in the intracellular environment and provides novel ideas for the treatment of related diseases and drug development.

SNORA70E promotes the occurrence and development of ovarian cancer through pseudouridylation modification of RAP1B and alternative splicing of PARPBP.

The present study demonstrated for the first time that SNORA70E, which belongs to box H/ACA small nucleolar noncoding RNAs (snoRNAs) who could bind and induce pseudouridylation of RNAs, was significantly elevated in ovarian cancer tissues and was an unfavourable prognostic factor of ovarian cancer. The over-expression of SNORA70E showed increased cell proliferation, invasion and migration in vitro and induced tumour growth in vivo. Further research found that SNORA70E regulates RAS-Related Protein 1B (RAP1B) mRNA through pseudouracil modification by combing with the pyrimidine synthase Dyskerin Pseudouridine Synthase 1 (DKC1) and increase RAP1B protein level. What's more, the silencing of DKC1/RAP1B in SNORA70E overexpression cells both inhibited cell proliferation, migration and invasion through reducing ß-catenin, PI3K, AKT1, mTOR, and MMP9 protein levels. Besides, RNA-Seq results revealed that SNORA70E regulates the alternative splicing of PARP-1 binding protein (PARPBP), leading to the 4th exon-skipping in PARPBP-88, forming a new transcript PARPBP-15, which promoted cell invasion, migration and proliferation. Finally, ASO-mediated silencing of SNORA70E could inhibit ovarian cancer cell proliferation, invasion, migration ability in vitro and inhibit tumorigenicity in vivo. In conclusion, SNORA70E promotes the occurrence and development of ovarian cancer through pseudouridylation modification of RAP1B and alternative splicing of PARPBP. Our results demonstrated that SNORA70E may be a new diagnostic and therapeutic target for ovarian cancer.

hsa-miR-23a~27a~24-2 cluster members inhibit aggressiveness of breast cancer cells by commonly targeting NCOA1, NLK and RAP1B.

Targeted therapy is receiving considerable attention from the researchers around the globe owing to the increased drug-resistance and incidences of cancer recurrences. MicroRNAs (miRNAs) exhibits tremendous potential as a candidate for molecular targeted therapy in cancer. Unfortunately, majority of research related to microRNAs are focussed on either a particular miRNA or a set of unrelated miRNAs. There is lack of holistic knowledge on differential co-expression of miRNA clusters in regulating the gene expression under physiological conditions. Previously, we reported the cooperative effect of hsa-miR-23a~27a~24-2 cluster in inducing ER (Endoplasmic Reticulum) stress-mediated apoptotic cell death of HEK cells. In the present study, we have investigated the common anti-cancer effects of individual members of this cluster. Our in silico analysis identified twelve common target genes distributed across three independent clusters. Furthermore, we found NCOA1, NLK, and RAP1B to fall in a single cluster with NCOA1 as a central hub molecule. Prognostic analysis showed profound involvement of these three genes in the breast cancer progression and metastasis. We further demonstrated that alteration in the levels of individual members of miR-23a~27a~24-2 cluster commonly regulates the invasive migration of breast cancer cells by modulating EMT and cytoskeletal pathway proteins. Our results reveal a new insight into the therapeutic potential of individual members of the pro-apoptotic hsa-miR-23a~27a~24-2 cluster family against metastatic breast cancer.

Evidence for a PI3-kinase independent pathway in the regulation of Rap1b activation downstream of the P2Y12 receptor in platelets.

Platelet activation by adenosine diphosphate (ADP) is mediated through two G-protein-coupled receptors, P2Y1 and P2Y12, which signal through Gq and Gi, respectively. P2Y1 stimulation leads to phospholipase C activation and an increase in cytosolic calcium necessary for CalDAG-GEF1 activation. Engagement of P2Y12 inhibits adenylate cyclase, which reduces cAMP, and activation of PI3-kinase, which inhibits RASA3 resulting in sustained activated Rap1b. In this study we activated human platelets with 2-MeSADP in the presence of LY294002, a PI3-kinase inhibitor, AR-C69931MX, a P2Y12 antagonist or MRS2179, a P2Y1 antagonist. We measured the phosphorylation of Akt on Ser473 as an indicator of PI3-kinase activity. As previously shown, LY294002 and ARC69931MX abolished 2MeSADP-induced Akt phosphorylation. MRS2179 reduced ADP-induced Akt phosphorylation but did not abolish it. Rap1b activation, however, was only reduced, but not ablated, using LY294002 and was completely inhibited by ARC69931MX or MRS2179. Furthermore, 2MeSADP-induced Rap1b activation was abolished in either P2Y1 or P2Y12 null platelets. These data suggest that ADP-induced Rap1b activation requires both P2Y1 and P2Y12. In addition, although stimulation of P2Y12 results in PI3-kinase activation leading to Akt phosphorylation and Rap1b activation, Rap1b activation can occur independently of PI3-kinase downstream of P2Y12. Thus, we propose that the P2Y12 receptor can regulate Rap1b, possibly through RASA3, in a pathway independent of PI3-kinase.

The E6 Oncoprotein of HPV16 AA-c Variant Regulates Cell Migration through the MINCR/miR-28-5p/RAP1B Axis.

The E6 oncoprotein of HPV16 variants differentially alters the transcription of the genes involved in migration and non-coding RNAs such as lncRNAs. The role of the lncRNA MINCR in cervical cancer and its relationship with variants of oncogenic HPV remain unknown. Therefore, the objective of this study was to analyze the effect of the E6 oncoprotein of the AA-c variant of HPV16 in cell migration through the MINCR/miR-28-5p/RAP1B axis. To explore the functional role of MINCR in CC, we used an in vitro model of C33-A cells with exogenous expression of the E6 oncoprotein of the AA-c variant of HPV16. Interfering RNAs performed MINCR silencing, and the expression of miR-28-5p and RAP1B mRNA was analyzed by RT-qPCR. We found that C33-A/AA-c cells expressed MINCR 8-fold higher compared to the control cells. There is an inverse correlation between the expression of miR-28-5p and RAP1B in C33-A/AA-c cells. Our results suggest that MINCR might regulate the expression of RAP1B through the inhibition of miR-28-5p in CC cells expressing the E6 oncoprotein of HPV16 AA-c. We report, for the first time, that the MINCR/miR-28-5p/RAP1B axis positively regulates cell migration in CC-derived cells that express the E6 oncoprotein of the AA-c variant of HPV16.

Third reported patient with RAP1B-related syndromic thrombocytopenia and novel clinical findings.

RAP1B is a RAS-superfamily small GTP-binding protein involved in numerous cell processes. Pathogenic gain-of-function variants in this gene have been associated with RAP1B-related syndromic thrombocytopenia, an ultrarare disorder characterized by hematologic abnormalities, neurodevelopmental delays, growth delay, and congenital birth defects including cardiovascular, genitourinary, neurologic, and skeletal systems. We report a 23-year-old male with a novel, de novo RAP1B gain-of-function variant identified on genome sequencing. This is the third reported case which expands the molecular and phenotypic spectrum of RAP1B-related syndromic thrombocytopenia.

Abnormal phenotype of Nrf2 is associated with poor prognosis through hypoxic/VEGF-A-Rap1b/VEGFR2 pathway in gastric cancer.

Metastasis is the major cause of death in gastric cancer patients and altered expression of Nrf2 is associated with cancer development. This study assessed Nrf2 and HO-1 expression and hypoxia-induced Nrf2 expression in the promotion of metastatic potential of gastric cancer cells, the relationship of Rap1b and Nrf2 was also discussed. Nrf2 and HO-1 expression were significantly associated with clinicopathological characteristic and were independent prognostic predictors in gastric cancer patients. Hypoxia up-regulated the expression of Nrf2, HO-1 and HIF-1α, whereas knockdown of Nrf2 inhibited cell invasion capacity and reduced the expression of Nrf2, HO-1 and HIF-1α. Patients in the Rap1b (+) Nrf2 (+) group had worst overall survival compared with those from other groups. Knockdown of Rap1b and Nrf2 significantly inhibited cell invasion capacity in the common group compared with the other groups. Hypoxia or VEGF-A facilitated the nuclear translocation of Nrf2 through Rap1b or VEGFR2. Hypoxia or VEGF-A did not induce the phosphorylation of P-Erk1/2 and P-Akt after knockdown of Rap1b or VEGFR2. Hypoxia promoted the gastric cancer malignant behavior through the upregulation of Rap1b and Nrf2. Hypoxia/VEGF-A-Rap1b/VEGFR2 facilitated the nuclear translocation of Nrf2. Targeting Rap1b and Nrf2 may be a novel therapeutic strategy for gastric cancer.

Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development.

Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins abbreviated as RAP-1a and RAP-1b. The full-length RAP-1a protein was expressed to perform the analysis but our efforts to express the full-length RAP-1b protein failed due to an unknown reason. We therefore generated synthetic immunogenic peptides that map onto the N- and C-termini of the RAP-1b protein as an alternative approach. Our findings show that both proteins are expressed in the extracellular and intra-erythrocytic merozoite stages of T. equi. Serological analyses show that T. equi-infected horses mount antibody responses that recognise both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralisation studies show that the T. equi RAP-1a protein contains neutralisation-sensitive epitopes as antibodies developed against the protein significantly inhibited the parasites from invading equine erythrocytes. Conversely, antibodies developed against the RAP-1b synthetic peptides did not neutralise parasite invasion, showing that the protein regions on which the peptides were based are not required for T. equi invasion. Overall, the data shows that T. equi rhoptries and their contents are involved in invasion of host cells and supports T. equi RAP-1 proteins as candidates for developing novel serodiagnosis tools and vaccines.

Avian Hepatitis E Virus ORF2 Protein Interacts with Rap1b to Induce Cytoskeleton Rearrangement That Facilitates Virus Internalization.

Avian hepatitis E virus (HEV) causes liver diseases and multiple extrahepatic disorders in chickens. However, the mechanisms involved in avian HEV entry remain elusive. Herein, we identified the RAS-related protein 1b (Rap1b) as a potential HEV-ORF2 protein interacting candidate. Experimental infection of chickens and cells with an avian HEV isolate from China (CaHEV) led to upregulated expression and activation of Rap1b both in vivo and in vitro. By using CaHEV capsid as mimic of virion to treat cell in vitro, it appears that the interaction between the viral capsid and Rap1b promoted cell membrane recruitment of the downstream effector Rap1-interacting molecule (RIAM). In turn, RIAM further enhanced Talin-1 membrane recruitment and retention, which led to the activation of integrin α5/ß1, as well as integrin-associated membrane protein kinases, including focal adhesion kinase (FAK). Meanwhile, FAK activation triggered activation of downstream signaling molecules, such as Ras-related C3 botulinum toxin substrate 1 RAC1 cell division cycle 42 (CDC42), p21-activated kinase 1 (PAK1), and LIM domain kinase 1 (LIMK1). Finally, F-actin rearrangement induced by Cofilin led to the formation of lamellipodia, filopodia, and stress fibers, contributes to plasma membrane remodeling, and might enhance CaHEV virion internalization. In conclusion, our data suggested that Rap1b activation was triggered during CaHEV infection and appeared to require interaction between CaHEV-ORF2 and Rap1b, thereby further inducing membrane recruitment of Talin-1. Membrane-bound Talin-1 then activates key Integrin-FAK-Cofilin cascades involved in modulation of actin kinetics, and finally leads to F-actin rearrangement and membrane remodeling to potentially facilitate internalization of CaHEV virions into permissive cells. IMPORTANCE Rap1b is a multifunctional protein that is responsible for cell adhesion, growth, and differentiation. The inactive form of Rap1b is phosphorylated and distributed in the cytoplasm, while active Rap1b is prenylated and loaded with GTP to the cell membrane. In this study, the activation of Rap1b was induced during the early stage of avian HEV infection under the regulation of PKA and SmgGDS. Continuously activated Rap1b recruited its effector RIAM to the membrane, thereby inducing the membrane recruitment of Talin-1 that led to the activation of membrane α5/ß1 integrins. The triggering of the signaling pathway-associated Integrin α5/ß1-FAK-CDC42&RAC1-PAK1-LIMK1-Cofilin culminated in F-actin polymerization and membrane remodeling that might promote avian HEV virion internalization. These findings suggested a novel mechanism that is potentially utilized by avian HEV to invade susceptible cells.

DF-1 cells prevent MG-HS infection through gga-miR-24-3p/RAP1B mediated decreased proliferation and increased apoptosis.

Mycoplasma gallisepticum (MG) is a major poultry pathogen that can induce Chronic Respiratory Disease (CRD) in chickens, causing serious economic losses in the poultry industry worldwide. Increasing evidence suggests that microRNAs (miRNAs) act as a vital role in resisting microbial pathogenesis and maintaining cellular mechanism. Our previous miRNAs sequencing data showed gga-miR-24-3p expression level was significantly increased in MG-infected chicken lungs. The aim of this study is to reveal the cellular mechanism behind the MG-HS infection. We found that gga-miR-24-3p was significantly upregulated and Ras-related protein-B (RAP1B) was downregulated in chicken fibroblast cells (DF-1) with MG infection. Dual luciferase reporting assay and rescue assay confirmed that RAP1B was the target gene of gga-miR-24-3p. Meanwhile, overexpressed gga-miR-24-3p increased the levels of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß), and significantly inhibited cell proliferation as well as promoted MG-infected DF-1 cell apoptosis, whereas inhibition of gga-miR-24-3p had the opposite effect. More importantly, the results of overexpression and knockdown of target gene RAP1B demonstrated that the presence of RAP1B promoted cell proliferation and it saved the reduced or increased cell proliferation caused by overexpression or inhibition of gga-miR-24-3p. Furthermore, the overexpression of gga-miR-24-3p could significantly inhibit the expression of MG-HS adhesion protein. Taken together, these findings demonstrate that DF-1 cells can resist MG-HS infection through gga-miR-24-3p/RAP1B mediated decreased proliferation and increased apoptosis, which provides a new mechanism of resistance to MG infection in vitro.

Corrigendum: Distinct Signaling Functions of Rap1 Isoforms in NO Release From Endothelium.

[This corrects the article DOI: 10.3389/fcell.2021.687598.].