Ras-related Proteins Research Articles

Identification, characterization and functional analysis of Rap1 in response to bacterial infection in tilapia

Rap1 (Ras-related protein 1), as a member of the Ras-like small GTP binding protein family, involves in the regulation of cell life activities and mediates a variety of biological events including cell junctions, cell polarity, cell proliferation and differentiation by binding to GDP/GTP. In addition, Rap1 also plays an important role in innate immune response by regulating the adhesion, diffusion and migration of white blood cells. However, it is still unclear how Rap1 affects the innate immunity of teleost fish against bacterial infections. In this study, we amplified the Rap1 from tilapia and conducted the sequence analysis of Rap1 nucleotide and amino acid sequences, then characterized its tissue distribution and expression pattern in response to pathogen infection in tilapia for the first time, and explored the bacteriostatic and bacterial binding effects of Rap1. The results showed that the Rap1 possessed a whole open reading frame (ORF) with 558 bp, encoding 185 amino acids. The Rap1 protein was highly homologous with Rap1 from other species. Conserved domain analysis revealed that Rap1 contained five G box domains that mediated its binding to ornithine. Rap1 mRNA was widely expressed in tilapia tissues, and the highest level was found in spleen. Under Streptococcus agalactiae (S. agalactiae) infection, the expression of Rap1 was significantly up-regulated in head kidney and brain. In addition, the recombinant protein Rap1 hold the ability binding to S. agalactiae and inhibit the proliferation of S. agalactiae. In conclusion, this study not only provided a reference for exploring the function of Rap1 in teleost against pathogen infection, but also enriched the basic theoretical research of Rap1 function and antibacterial immune response in teleost.

Telomeres cooperate with the nuclear envelope to maintain genome stability.

Mammalian telomeres have evolved safeguards to prevent their recognition as DNA double-stranded breaks by suppressing the activation of various DNA sensing and repair proteins. We have shown that the telomere-binding proteins TRF2 and RAP1 cooperate to prevent telomeres from undergoing aberrant homology-directed recombination by mediating t-loop protection. Our recent findings also suggest that mammalian telomere-binding proteins interact with the nuclear envelope to maintain chromosome stability. RAP1 interacts with nuclear lamins through KU70/KU80, and disruption of RAP1 and TRF2 function result in nuclear envelope rupture, promoting telomere-telomere recombination to form structures termed ultrabright telomeres. In this review, we discuss the importance of the interactions between shelterin components and the nuclear envelope to maintain telomere homeostasis and genome stability.

Context-dependent function of the transcriptional regulator Rap1 in gene silencing and activation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, heterochromatin is formed through interactions between site-specific DNA-binding factors, including the transcriptional activator Repressor Activator Protein (Rap1), and Sir proteins. Despite an understanding of the establishment and maintenance of Sir-silenced chromatin, the mechanism of gene silencing by Sir proteins has remained a mystery. Utilizing high-resolution chromatin immunoprecipitation, we found that Rap1, the native activator of the bidirectional HMLα promoter, bound its recognition sequence in silenced chromatin, and its binding was enhanced by the presence of Sir proteins. In contrast to prior results, various components of transcription machinery were not able to access HMLα in the silenced state. These findings disproved the long-standing model of indiscriminate steric occlusion by Sir proteins and led to investigation of the role of the transcriptional activator Rap1 in Sir-silenced chromatin. Using a highly sensitive assay that monitors loss-of-silencing events, we identified a role for promoter-bound Rap1 in the maintenance of silent chromatin through interactions with the Sir complex. We also found that promoter-bound Rap1 activated HMLα when in an expressed state, and aided in the transition from transcription initiation to elongation. Highlighting the importance of epigenetic context in transcription factor function, these results point toward a model in which the duality of Rap1 function was mediated by local chromatin environment rather than binding-site availability.

Crucial roles of Rab22a in endosomal cargo recycling.

Endosomal cargo recycling lies at the heart of subcellular trafficking processes under the management of several Ras-related GTP-binding proteins (Rabs) which are coordinated by their upstream regulators and require their downstream effectors to display their functions. In this regard, several Rabs have been well-reviewed except Rab22a. Rab22a is a crucial regulator of vesicle trafficking, early endosome and recycling endosome formation. Notably, recent studies demonstrated the immunological roles of Rab22a, which are closely associated with cancers, infection and autoimmune disorders. This review provides an overview of the regulators and effectors of Rab22a. Also, we highlight the current knowledge of the role of Rab22a in endosomal cargo recycling, including the biogenesis of recycling tubules with the help of a complex with Rab22a at its core, and how different internalized cargo chooses different recycling routes thanks to the cooperation of Rab22a, its effectors and its regulators. Of note, contradictions and speculation related to endosomal cargo recycling that Rab22a brings impacts on are also discussed. Finally, this review endeavors to briefly introduce the various events impacted by Rab22a, particularly focusing on the commandeered Rab22a-associated endosomal maturation and endosomal cargo recycling, in addition to the extensively investigated oncogenic role of Rab22a.

476-P: Analysis of Upstream Signaling Pathway of Erk2 following Transient High-Glucose Exposure in Coronary Artery Endothelial Cells

Using an experimental approach, we investigated the mechanisms by which brief high-glucose exposure activates extracellular signal-regulated kinase 2 (Erk2) in cultured human coronary artery endothelial cells. The cells were cultured in standard medium for 14 days (5.5 mM D-glucose). Thereafter, transient glucose elevation was achieved by adding either 183 mg/dL L-glucose or 183 mg/dL D-glucose to the culture medium for 1 h. The Erk2 activation status was measured by western blot analysis of phospho-Erk2. As part of the analysis of the upstream signaling pathway of Erk2, Rap1 and Ras activation status were examined. Furthermore, guanine nucleotide-binding protein G (i) α2 (Giα2) protein and microRNA-138 (miR-138) levels were measured. Ras and Erk2 were activated, whereas Rap1 was deactivated following addition of 183 mg/dL D-glucose to the culture medium and incubation for 1 h. Furthermore, Giα2 protein levels increased and miR-138 levels decreased. The addition of 183 mg/dL L-glucose did not affect the results. In general, our findings suggest that transient exposure to high amounts of D-glucose decreased miR-138 levels, resulting in an increase in Giα2 protein levels, Rap1 deactivation, Ras activation, and Erk2 activation. Here we report a novel mechanism that links transient high-glucose levels to Erk2 activation in cultured human coronary artery endothelial cells. Disclosure J.Okada: None. E.Yamada: None. T.Saito: None. S.Okada: None.

Telomere loss is accompanied by decreased pool of shelterin proteins TRF2 and RAP1, elevated levels of TERRA and enhanced glycolysis in imatinib-resistant CML cells

Telomere length may be maintained by telomerase nucleoprotein complex and shelterin complex, namely TRF1, TRF2, TIN2, TPP1, POT1 and RAP1 proteins and modulated by TERRA expression. Telomere loss is observed during progression of chronic myeloid leukemia (CML) from the chronic phase (CML-CP) to the blastic phase (CML-BP). The introduction of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), has changed outcome for majority of patients, however, a number of patients treated with TKIs may develop drug resistance. The molecular mechanisms underlying this phenomenon are not fully understood and require further investigation. In the present study, we demonstrate that IM-resistant BCR::ABL1 gene-positive CML K-562 and MEG-A2 cells are characterized by decreased telomere length, lowered protein levels of TRF2 and RAP1 and increased expression of TERRA in comparison to corresponding IM-sensitive CML cells and BCR::ABL1 gene-negative HL-60 cells. Furthermore, enhanced activity of glycolytic pathway was observed in IM-resistant CML cells. A negative correlation between a telomere length and advanced glycation end products (AGE) was also revealed in CD34+ cells isolated from CML patients. In conclusion, we suggest that affected expression of shelterin complex proteins, namely TRF2 and RAP1, TERRA levels, and glucose consumption rate may promote telomere dysfunction in IM-resistant CML cells.

A comprehensive in vivo screen of yeast farnesyltransferase activity reveals broad reactivity across a majority of CXXX sequences.

The current understanding of farnesyltransferase (FTase) specificity was pioneered through investigations of reporters like Ras and Ras-related proteins that possess a C-terminal CaaX motif that consists of 4 amino acid residues: cysteine-aliphatic1-aliphatic2-variable (X). These studies led to the finding that proteins with the CaaX motif are subject to a 3-step post-translational modification pathway involving farnesylation, proteolysis, and carboxylmethylation. Emerging evidence indicates, however, that FTase can farnesylate sequences outside the CaaX motif and that these sequences do not undergo the canonical 3-step pathway. In this work, we report a comprehensive evaluation of all possible CXXX sequences as FTase targets using the reporter Ydj1, an Hsp40 chaperone that only requires farnesylation for its activity. Our genetic and high-throughput sequencing approach reveals an unprecedented profile of sequences that yeast FTase can recognize in vivo, which effectively expands the potential target space of FTase within the yeast proteome. We also document that yeast FTase specificity is majorly influenced by restrictive amino acids at a2 and X positions as opposed to the resemblance of CaaX motif as previously regarded. This first complete evaluation of CXXX space expands the complexity of protein isoprenylation and marks a key step forward in understanding the potential scope of targets for this isoprenylation pathway.

The human RAP1 and GFAPɛ proteins increase γ-secretase activity in a yeast model system.

Alzheimer's disease (AD) is an age-related disorder that results in progressive cognitive impairment and memory loss. Deposition of amyloid β (Aβ) peptides in senile plaques is a hallmark of AD. γ-secretase produces Aβ peptides, mostly as the soluble Aβ40 with fewer insoluble Aβ42 peptides. Rare, early-onset AD (EOAD) occurs in individuals under 60 years of age. Most EOAD cases are due to unknown genetic causes, but a subset is due to mutations in the genes encoding the amyloid precursor protein that is processed into Aβ peptides or the presenilins (PS1 and PS2) that process APP. PS1 interacts with the epsilon isoform of glial fibrillary acidic protein (GFAPɛ), a protein found in the subventricular zone of the brain. We have found that GFAPɛ interacts with the telomere protection factor RAP1 (TERF2IP). RAP1 can also interact with PS1 alone or with GFAPɛ in vitro. Our data show that the nuclear protein RAP1 has an extratelomeric role in the cytoplasm through its interactions with GFAPɛ and PS1. GFAPɛ coprecipitated with RAP1 from human cell extracts. RAP1, GFAPɛ, and PS1 all colocalized in human SH-SY5Y cells. Using a genetic model of the γ-secretase complex in Saccharomyces cerevisiae, RAP1 increased γ-secretase activity, and this was potentiated by GFAPɛ. Our studies are the first to connect RAP1 with an age-related disorder.

CAMP-Epac1 signaling is activated in DDAVP-induced endolymphatic hydrops of guinea pigs

ObjectiveTo explore whether Cyclic Adenosine Monophosphate (cAMP)-Epac1 signaling is activated in 1-Desamino-8-D-arginine-Vasopressin-induced Endolymphatic Hydrops (DDAVP-induced EH) and to provide new insight for further in-depth study of DDAVP-induced EH. MethodsEighteen healthy, red-eyed guinea pigs (36 ears) weighing 200–350 g were randomly divided into three groups: the control group, which received intraperitoneal injection of sterile saline (same volume as that in the other two groups) for 7 consecutive days; the DDAVP-7d group, which received intraperitoneal injection of 10 mg/mL/kg DDAVP for 7 consecutive days; and the DDAVP-14d group, which received intraperitoneal injection of 10 μg/mL/kg DDAVP for 14 consecutive days. After successful modeling, all animals were sacrificed, and cochlea tissues were collected to detect the mRNA and protein expression of the exchange protein directly activated by cAMP-1 and 2 (Epac1, Epac2), and Repressor Activator Protein-1 (Rap1) by Reverse Transcription (RT)-PCR and western blotting, respectively. ResultsCompared to the control group, the relative mRNA expression of Epac1, Epac2, Rap1A, and Rap1B in the cochlea tissue of the DDAVP-7d group was significantly higher (p < 0.05), while no significant difference in Rap1 GTPase activating protein (Rap1gap) mRNA expression was found between the two groups. The relative mRNA expression of Epac1, Rap1A, Rap1B, and Rap1gap in the cochlea tissue of the DDAVP-14d group was significantly higher than that of the control group (p < 0.05), while no significant difference in Epac2 mRNA expression was found between the DDAVP-14d and control groups. Comparison between the DDAVP-14d and DDAVP-7d groups showed that the DDAVP-14d group had significantly lower Epac2 and Rap1A (p < 0.05) and higher Rap1gap (p < 0.05) mRNA expression in the cochlea tissue than that of the DDAVP-7d group, while no significant differences in Epac1 and Rap1B mRNA expression were found between the two groups. Western blotting showed that Epac1 protein expression in the cochlea tissue was the highest in the DDAVP-14d group, followed by that in the DDAVP-7d group, and was the lowest in the control group, showing significant differences between groups (p < 0.05); Rap1 protein expression in the cochlea tissue was the highest in the DDAVP-7d group, followed by the DDAVP-14d group, and was the lowest in the control group, showing significant differences between groups (p < 0.05); no significant differences in Epac2 protein expression in the cochlea tissue were found among the three groups. ConclusionDDAVP upregulated Epac1 protein expression in the guinea pig cochlea, leading to activation of the inner ear cAMP-Epac1 signaling pathway. This may be an important mechanism by which DDAVP regulates endolymphatic metabolism to induce EH and affect inner ear function. Oxford Centre for Evidence-Based Medicine 2011 Levels of EvidenceLevel 5.

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.

Small GTP-binding protein Rap1 mediates EGF and HB-EGF signaling and modulates EGF receptor expression in HTR-8/SVneo extravillous trophoblast cells.

Extravillous trophoblasts (EVTs) invade the endometrium to establish a fetomaternal interaction during pregnancy. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) stimulate EVT invasion by binding to the EGF receptor (EGFR). We examined the role of the small GTP-binding protein Rap1 in EGF- and HB-EGF-stimulated EVT invasion. Expression of Rap1 in the first-trimester placenta was examined by immunohistochemistry. Effect of EGF or HB-EGF on Rap1 activation (GTP-Rap1) and Rap1 knockdown on invasion was assessed in EVT cell line (HTR-8/SVneo). In addition, effect of Rap1 knockdown and Rap1GAP (a Rap1 inactivator) overexpression on the activation of EGF signaling and EGFR expression were examined. Rap1 was expressed by EVTs, villous cytotrophoblasts, and syncytiotrophoblasts in the placenta. EGF and HB-EGF activated Rap1 and promoted invasion of HTR-8/SVneo, and these effects were inhibited by Rap1 knockdown. The EGF- and HB-EGF-induced phosphorylation of AKT, ERK1/2, p38MAPK, and Src was inhibited by Rap1 knockdown. Furthermore, the knockdown of Rap1 reduced the EGFR protein level. Overexpression of Rap1GAP repressed EGF- and HB-EGF-induced Rap1 activation and reduced EGFR expression. Rap1 may function as a mediator of EGF and HB-EGF signaling pathways and can modulate EGFR expression in EVTs during placental development.

Synovial Fluid in Knee Osteoarthritis Extends Proinflammatory Niche for Macrophage Polarization.

Macrophage polarization is a steering factor of osteoarthritis (OA) progression. Synovial fluid (SF) obtained from OA patients with different Kellgren-Lawrence grades (KL grades) holds several proinflammatory factors and was hypothesized to induce macrophage differentiation and polarization by providing the needed microenvironment. U937 cells and peripheral-blood-mononuclear-cell-derived monocytes (PBMC-derived CD14+ cells) were induced with SFs of progressive KL grades for 48 h, and the status of the differentiated cells was evaluated by cell surface markers representing M1 and M2 macrophage phenotypes. Functional viability assessment of the differentiated cells was performed by cytokine estimation. The fraction of macrophages and their phenotypes were estimated by immunophenotyping of SF-isolated cells of different KL grades. A grade-wise proteome analysis of SFs was performed in search of the factors which are influential in macrophage differentiation and polarization. In the assay on U937 cells, induction with SF of KL grade III and IV showed a significant increase in M1 type (CD86+). The percentage of M2 phenotype (CD163+) was significantly higher after the induction with SF of KL grade II. A Significantly higher M1/M2 ratio was estimated in the cells induced with KL grade III and IV. The cell differentiation pattern in the assay on PBMC-derived CD14+ cells showed a grade-wise decline in both M1 (CD11C+, CD86+) and M2 phenotype (CD163+). Cytokine estimation specific to M1 (TNF-α, IL-6, IL-1β, IFN-γ) and M2 (IL-4 and IL-10) macrophages corelated with the differentiation pattern in the U937 cell assay, while it did not reveal any significant changes in the PBMC-derived CD14+ cells assay. SF cells' immunophenotyping showed the highest percentage of CD14+ macrophages in KL grade II; CD86+ and CD163+ cells were minimal in all KL grades' SFs. The proteome analysis revealed significantly expressed MIF, CAPG/MCP, osteopontin, and RAS-related RAB proteins in KL grade III and IV samples, which are linked with macrophages' movement, polarization, and migration-behavior. In conclusion, this study demonstrated that SF in OA joints acts as a niche and facilitates M1 phenotype polarization by providing a proinflammatory microenvironment.

Prediction of male fertility using Ras-related proteins

Identifying effective biomarkers for the diagnosis of male fertility is crucial for improving animal production and treating male infertility in humans. Ras-related proteins (Rab) are associated with morphological and motion kinematic functions in spermatozoa. Moreover, Rab2A, a Rab protein, is a possible male fertility-related biomarker. The present study was designed to identify additional fertility-related biomarkers among the various Rab proteins. First, the expression of Rab proteins (Rab3A, 4, 5, 8A, 9, 14, 25, 27A, and 34A) from 31 duroc boar spermatozoa was measured before and after capacitation; correlation between Rab protein expression and litter size was evaluated by statistical analysis. The results showed that the expression of Rab3A, 4, 5, 8A, 9, and 25 before capacitation and Rab3A, 4, 5, 8A, 9, and 14 after capacitation were negatively correlated with litter size. Moreover, depending on the cut-off values calculated by receiver operating curves, an increase in litter size was observed when evaluating the ability of the Rab proteins to forecast litter size. Therefore, we suggest that Rab proteins may be potential fertility-related biomarkers that could help select superior sires in the livestock industry.

Rap1 prevents fusions between long telomeres in fission yeast

The conserved Rap1 protein is part of the shelterin complex that plays critical roles in chromosome end protection and telomere length regulation. Previous studies have addressed how fission yeast Rap1 contributes to telomere length maintenance, but the mechanism by which the protein inhibits end fusions has remained elusive. Here, we use a mutagenesis screen in combination with high‐throughput sequencing to identify several amino acid positions in Rap1 that have key roles in end protection. Interestingly, mutations at these sites render cells susceptible to genome instability in a conditional manner, whereby longer telomeres are prone to undergoing end fusions, while telomeres within the normal length range are sufficiently protected. The protection of long telomeres is in part dependent on their nuclear envelope attachment mediated by the Rap1–Bqt4 interaction. Our data demonstrate that long telomeres represent a challenge for the maintenance of genome integrity, thereby providing an explanation for species‐specific upper limits on telomere length.

The effects of different exercise modes on Rab5 protein and glucose metabolism in skeletal muscle of type 2 diabetic mellitus rats

Objective: To investigate the effects of continuing exercise and load-bearing interval exercise on skeletal muscle tissue cell morphology, Ras-related proteins 5 (Rab5) mRNA and protein expression and glucose metabolism in skeletal muscle of type 2 diabetic mellitus (T2DM) rats. Methods: Eight SD rats were selected as controls group (CR), the others SD rats were fed with high fat and high sugar diet for 6 weeks before injecting STZ (35 mg/kg) to construct the T2DM model. Twenty-four T2DM rats were randomly devided into T2DM model group (DRM), continuing exercise group (DCRE) and load-bearing interval exercise group (DWRE), 8 rats in each group. DCRE exercise protocol, that was 15 m/min (10 min), 20 m/min (40 min), 15 m/min (10 min), during the first 1～2 weeks, and 18 m/min (10 min), 25 m/min (40 min), 15 m/min (10 min), during the second 3～8 weeks. DWRE exercise protocol: load weight 15% / 1～2 weeks, 30% / 3～4 weeks, 45% / 5～8 weeks, with 15 m/min (5 min), 12 groups and 3 min rest between groups. After 8 weeks, pathological and morphological changes of skeletal muscle were observed by HE. Rab5 and Glucose transporte 4 (GLUT4) mRNA expressions of skeletal muscle were tested by qRT-PCR. Rab5 protein expression in skeletal muscle was tested by immunofluorescence histochemistry and Western blot, and plasma Rab5 and Glycosylated Hemoglobin (GHb) concentrations were detected by ELISA. Results: Comparison with CR, DRM showed pathological damage of skeletal muscle, the expressions of Rab5 mRNA, protein and GLUT4 mRNA were all decreased in skeletal muscle (P＜0.01), the serum levels of Rab5 and GHb were both significantly elevated (P＜0.01). Comparison with DRM, both DCRE and DWRE significantly improved pathological damages of skeletal muscle, the expressions of Rab5 mRNA, protein and GLUT4 mRNA were all increased in skeletal muscle (P＜ 0.05, P＜0.01), the serum levels of Rab5 and GHb were decreased (P＜0.05, P＜0.01), and there was no statistical difference between DCRE and DWRE groups (P＞0.05). Conclusion: Two exercise modes can improve the pathological injury of skeletal muscle in type 2 diabetic rats, and enhance GLUT4 transport capacity by improving the expression of Rab5 gene and protein in skeletal muscle, and alleviate the imbalance of glucose metabolism homeostasis in skeletal muscle. However, there was no significant difference between the effects of two exercise modes on Rab5 protein and glucose metabolism in skeletal muscle.

A CRISPR screen targeting PI3K effectors identifies RASA3 as a negative regulator of LFA-1-mediated adhesion in T cells.

The integrin lymphocyte function-associated antigen 1 (LFA-1) helps to coordinate the migration, adhesion, and activation of T cells through interactions with intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. LFA-1 is activated during the engagement of chemokine receptors and the T cell receptor (TCR) through inside-out signaling, a process that is partially mediated by phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3). To evaluate potential roles of PI3K in LFA-1 activation, we designed a library of CRISPR/single guide RNAs targeting known and potential PIP3-binding proteins and screened for effects on the ability of primary mouse T cells to bind to ICAM-1. We identified multiple proteins that regulated the binding of LFA-1 to ICAM-1, including the Rap1 and Ras GTPase-activating protein RASA3. We found that RASA3 suppressed LFA-1 activation in T cells, that its expression was rapidly reduced upon T cell activation, and that its activity was inhibited by PI3K. Loss of RASA3 in T cells led to increased Rap1 activation, defective lymph node entry and egress, and impaired responses to T-dependent immunization in mice. Our results reveal a critical role for RASA3 in T cell migration, homeostasis, and function.

RapD Is a Multimeric Calcium-Binding Protein That Interacts With the Rhizobium leguminosarum Biofilm Exopolysaccharide, Influencing the Polymer Lengths

Rhizobium leguminosarum synthesizes an acidic polysaccharide mostly secreted to the extracellular medium, known as exopolysaccharide (EPS) and partially retained on the bacterial surface as a capsular polysaccharide (CPS). Rap proteins, extracellular protein substrates of the PrsDE type I secretion system (TISS), share at least one Ra/CHDL (cadherin-like) domain and are involved in biofilm matrix development either through cleaving the polysaccharide by Ply glycanases or by altering the bacterial adhesive properties. It was shown that the absence or excess of extracellular RapA2 (a monomeric CPS calcium-binding lectin) alters the biofilm matrix’s properties. Here, we show evidence of the role of a new Rap protein, RapD, which comprises an N-terminal Ra/CHDL domain and a C-terminal region of unknown function. RapD was completely released to the extracellular medium and co-secreted with the other Rap proteins in a PrsDE-dependent manner. Furthermore, high levels of RapD secretion were found in biofilms under conditions that favor EPS production. Interestingly, size exclusion chromatography of the EPS produced by the ΔrapA2ΔrapD double mutant showed a profile of EPS molecules of smaller sizes than those of the single mutants and the wild type strain, suggesting that both RapA2 and RapD proteins influence EPS processing on the cell surface. Biophysical studies showed that calcium triggers proper folding and multimerization of recombinant RapD. Besides, further conformational changes were observed in the presence of EPS. Enzyme-Linked ImmunoSorbent Assay (ELISA) and Binding Inhibition Assays (BIA) indicated that RapD specifically binds the EPS and that galactose residues would be involved in this interaction. Taken together, these observations indicate that RapD is a biofilm matrix-associated multimeric protein that influences the properties of the EPS, the main structural component of the rhizobial biofilm.

Exploration of the Molecular Mechanism of Danzhi Xiaoyao Powder in Endometrial Cancer through Network Pharmacology.

Endometrial cancer (EC) is a common malignant tumor of the female reproductive system. Current treatments such as surgery and long-term hormone therapy are ineffective and have side effects. Danzhi Xiaoyao powder (DXP) can inhibit the growth of EC cells and induce apoptosis, but the pharmacological and molecular mechanisms of anticancer effects are still unclear. In this study, active components and potential targets of DXP were obtained from public databases. Protein effects and regulatory pathways of common targets were analyzed by protein-protein interaction (PPI), GO and KEGG. The results of network pharmacology showed that there are 87 common targets between EC and DXP. GO enrichment analysis showed that these targets were associated with response to oxidative stress, response to nutrient levels, hormone receptor binding and nuclear hormone receptor binding, etc. The results of KEGG analysis indicated that IL-17, TNF, PI3K/AKT, and RAS/RAF/MEK/ERK (ERK) signaling pathway were enriched in the anti-EC of DXP. Additionally, we cultured HEC-1B and KLE cells for validate experiments. DXP showed an inhibition of proliferation, migration, and cell cycle of both cells. Moreover, the expression of RAS, p-RAF, p-MEK, ERK, and p-ERK related proteins were downregulated. In conclusion, DXP might inhibit the proliferation of EC cells via apoptosis. Furthermore, DXP-induced inhibition of EC development might involve RAS/RAF/MEK/ERK pathway.

LFA1 Activation: Insights from a Single-Molecule Approach.

Integrin LFA1 is a cell adhesion receptor expressed exclusively in leukocytes, and plays crucial roles in lymphocyte trafficking, antigen recognition, and effector functions. Since the discovery that the adhesiveness of LFA1 can be dynamically changed upon stimulation, one challenge has been understanding how integrins are regulated by inside-out signaling coupled with macromolecular conformational changes, as well as ligand bindings that transduce signals from the extracellular domain to the cytoplasm in outside-in signaling. The small GTPase Rap1 and integrin adaptor proteins talin1 and kindlin-3 have been recognized as critical molecules for integrin activation. However, their cooperative regulation of integrin adhesiveness in lymphocytes requires further research. Recent advances in single-molecule imaging techniques have revealed dynamic molecular processes in real-time and provided insight into integrin activation in cellular environments. This review summarizes integrin regulation and discusses new findings regarding the bidirectionality of LFA1 activation and signaling processes in lymphocytes.

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.