Structural and functional characterization of the bacterial Cap3 enzyme in deconjugation and regulation of the cyclic dinucleotide transferase CD-NTase.

The Cyclic GMP-AMP synthase (cGAS) and cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes belong to the key components of the innate immune sensor system that generates cyclic dinucleotide molecules in response to danger signals. Recently, it was discovered that CD-NTase in bacteria can undergo conjugation to protein substrates via an E1/E2 enzyme-mediated process, resembling ubiquitin modification system. Subsequently, these CD-NTase conjugated molecules will be hydrolyzed by the Cap3 enzyme in the same gene cluster. However, the experimental structure of bacterial CD-NTase recognized by Cap3 is unknown. Here, we first determined the crystal structure of the Cap3 enzyme in complex with the C-terminal tail of CD-NTase. Our structural and enzymatic analysis revealed that the C-terminal tail of CD-NTase is both necessary and sufficient for the Cap3-mediated hydrolysis of CD-NTase from its substrates. Interestingly, we further observed that after the hydrolysis reaction, the terminal glycine residue of the CD-NTase C-terminal tail was sequentially removed by Cap3, indicating that Cap3 might play a role in quenching the CD-NTase conjugation reaction. Our work provides experimental evidence elucidating the interaction between Cap3 and CD-NTase, and suggests a potential role for Cap3 in the bacterial Cyclic-oligonucleotide-based anti-phage signaling system (CBASS).

CAP2 contributes to Parkinson's disease diagnosed by neutrophil extracellular trap-related immune activity.

Introduction: Neutrophil extracellular traps (NETs) constitute a crucial element of the immune system, and dysfunction in immune responses is implicated in the susceptibility and progression of Parkinson's disease (PD). Nevertheless, the mechanism connecting PD and NETs remains unclear. This study aims to uncover potential NETs-related immune biomarkers and elucidate their role in PD pathogenesis. Methods: Through differential gene analysis of PD and NETs in GSE7621 datasets, we identified two PD subtypes and explored potential biological pathways. Subsequently, using ClusterWGCNA, we pinpointed pertinent genes and developed clinical diagnostic models. We then optimized the chosen model and evaluated its association with immune infiltration. Validation was conducted using the GSE20163 dataset. Screening the single-cell dataset GSE132758 revealed cell populations associated with the identified gene. Results: Our findings identified XGB as the optimal diagnostic model, with CAP2 identified as a pivotal gene. The risk model effectively predicted overall diagnosis rates, demonstrating a robust correlation between infiltrating immune cells and genes related to the XGB model. Discussion: In conclusions, we identified PD subtypes and diagnostic genes associated with NETs, highlighting CAP2 as a pivotal gene. These findings have significant implications for understanding potential molecular mechanisms and treatments for PD.

Essential roles of RNA cap-proximal ribose methylation in mammalian embryonic development and fertility.

Eukaryotic RNA pol II transcripts are capped at the 5' end by the methylated guanosine (m7G) moiety. In higher eukaryotes, CMTR1 and CMTR2 catalyze cap-proximal ribose methylations on the first (cap1) and second (cap2) nucleotides, respectively. These modifications mark RNAs as "self," blocking the activation of the innate immune response pathway. Here, we show that loss of mouse Cmtr1 or Cmtr2 leads to embryonic lethality, with non-overlapping sets of transcripts being misregulated, but without activation of the interferon pathway. In contrast, Cmtr1 mutant adult mouse livers exhibit chronic activation of the interferon pathway, with multiple interferon-stimulated genes being expressed. Conditional deletion of Cmtr1 in the germline leads to infertility, while global translation is unaffected in the Cmtr1 mutant mouse liver and human cells. Thus, mammalian cap1 and cap2 modifications have essential roles in gene regulation beyond their role in helping cellular transcripts to evade the innate immune system.

Periviscerokinin (Cap2b; CAPA) receptor silencing in females of Rhipicephalus microplus reduces survival, weight and reproductive output.

BACKGROUND: The cattle fever tick, Rhipicephalus (Boophilus) microplus, is a vector of pathogens causative of babesiosis and anaplasmosis, both highly lethal bovine diseases that affect cattle worldwide. In Ecdysozoa, neuropeptides and their G-protein-coupled receptors play a critical integrative role in the regulation of all physiological processes. However, the physiological activity of many neuropeptides is still unknown in ticks. Periviscerokinins (CAP2b/PVKs) are neuropeptides associated with myotropic and diuretic activities in insects. These peptides have been identified only in a few tick species, such as Ixodes ricinus, Ixodes scapularis and R. microplus, and their cognate receptor only characterized for the last two. METHODS: Expression of the periviscerokinin receptor (Rhimi-CAP2bR) was investigated throughout the developmental stages of R. microplus and silenced by RNA interference (RNAi) in the females. In a first experiment, three double-stranded (ds) RNAs, named ds680-805, ds956-1109 and ds1102-1200, respectively, were tested in vivo. All three caused phenotypic effects, but only the last one was chosen for subsequent experiments. Resulting RNAi phenotypic variables were compared to those of negative controls, both non-injected and dsRNA beta-lactamase-injected ticks, and to positive controls injected with beta-actin dsRNA. Rhimi-CAP2bR silencing was verified by quantitative reverse-transcriptase PCR in whole females and dissected tissues. RESULTS: Rhimi-CAP2bR transcript expression was detected throughout all developmental stages. Rhimi-CAP2bR silencing was associated with increased female mortality, decreased weight of surviving females and of egg masses, a delayed egg incubation period and decreased egg hatching (P < 0.05). CONCLUSIONS: CAP2b/PVKs appear to be associated with the regulation of female feeding, reproduction and survival. Since the Rhimi-CAP2bR loss of function was detrimental to females, the discovery of antagonistic molecules of the CAP2b/PVK signaling system should cause similar effects. Our results point to this signaling system as a promising target for tick control.

miRNA mediated downregulation of cyclase-associated protein 1 (CAP1) is required for myoblast fusion.

Myoblast fusion is essential for the formation, growth, and regeneration of skeletal muscle, but the molecular mechanisms that govern fusion and myofiber formation remain poorly understood. Past studies have shown an important role of the actin cytoskeleton and actin regulators in myoblast fusion. The Cyclase-Associated Proteins (CAP) 1 and 2 recently emerged as critical regulators of actin treadmilling in higher eukaryotes including mammals. Whilst the role of CAP2 in skeletal muscle development and function is well characterized, involvement of CAP1 in this process remains elusive. Here we report that CAP1, plays a critical role in cytoskeletal remodeling during myoblast fusion and formation of myotubes. Cap1 mRNA and protein are expressed in both murine C2C12 and human LHCN-M2 myoblasts, but their abundance decreases during myogenic differentiation. Perturbing the temporally controlled expression of CAP1 by overexpression or CRISPR-Cas9 mediated knockout impaired actin rearrangement, myoblast alignment, expression of profusion molecules, differentiation into multinucleated myotubes, and myosin heavy chain expression. Endogenous Cap1 expression is post-transcriptionally downregulated during differentiation by canonical myomiRs miR-1, miR-133, and miR-206, which have conserved binding sites at the 3' UTR of the Cap1 mRNA. Deletion of the endogenous 3' UTR by CRISPR-Cas9 in C2C12 cells phenocopies overexpression of CAP1 by inhibiting myotube formation. Our findings implicates Cap1 and its myomiR-mediated downregulation in the myoblast fusion process and the generation of skeletal muscle.

Disease association of cyclase-associated protein (CAP): Lessons from gene-targeted mice and human genetic studies.

Cyclase-associated protein (CAP) is an actin binding protein that has been initially described as partner of the adenylyl cyclase in yeast. In all vertebrates and some invertebrate species, two orthologs, named CAP1 and CAP2, have been described. CAP1 and CAP2 are characterized by a similar multidomain structure, but different expression patterns. Several molecular studies clarified the biological function of the different CAP domains, and they shed light onto the mechanisms underlying CAP-dependent regulation of actin treadmilling. However, CAPs are crucial elements not only for the regulation of actin dynamics, but also for signal transduction pathways. During recent years, human genetic studies and the analysis of gene-targeted mice provided important novel insights into the physiological roles of CAPs and their involvement in the pathogenesis of several diseases. In the present review, we summarize and discuss recent progress in our understanding of CAPs' physiological functions, focusing on heart, skeletal muscle and central nervous system as well as their involvement in the mechanisms controlling metabolism. Remarkably, loss of CAPs or impairment of CAPs-dependent pathways can contribute to the pathogenesis of different diseases. Overall, these studies unraveled CAPs complexity highlighting their capability to orchestrate structural and signaling pathways in the cells.

Efficacy and biosafety assessment of neuropeptide CAPA analogues against the peach-potato aphid (Myzus persicae).

Insect CAPA neuropeptidesare considered to affect water and ion balance by mediating the physiological metabolism activities of the Malpighian tubules. In previous studies, the CAPA-PK analogue 1895 (2Abf-Suc-FGPRLamide) was reported to decrease aphid fitness when administered through microinjection or via topical application. However, a further statistically significant decrease in the fitness of aphids and an increased mortality could not be established with pairwise combinations of 1895 with other CAPA analogue. In this study, we assessed the topical application of new combinations of 1895 with five CAPA-PVK analogues on the fitness of aphids. We found that 1895 and CAPA-PVK analogue 2315 (ASG-[ß3 L]-VAFPRVamide) was statistically the most effective combination to control the peach potato aphid Myzus persicae nymphs via topical application, leading to 72% mortality. Additionally, the combination (1895+2315) was evaluated against a selection of beneficial insects, that is, a pollinator (Bombus terrestris) and three natural enemies (Chrysoperla carnea, Nasonia vitripennis, and Adalia bipunctata). We found no significant influence on food intake, weight increase, and survival for the pollinator and the three representative natural enemies. These results could facilitate to further establish and generate CAPA analogues as alternatives to broad spectrum and less friendly insecticides.

Functional Redundancy of Cyclase-Associated Proteins CAP1 and CAP2 in Differentiating Neurons.

Cyclase-associated proteins (CAPs) are evolutionary-conserved actin-binding proteins with crucial functions in regulating actin dynamics, the spatiotemporally controlled assembly and disassembly of actin filaments (F-actin). Mammals possess two family members (CAP1 and CAP2) with different expression patterns. Unlike most other tissues, both CAPs are expressed in the brain and present in hippocampal neurons. We recently reported crucial roles for CAP1 in growth cone function, neuron differentiation, and neuron connectivity in the mouse brain. Instead, CAP2 controls dendritic spine morphology and synaptic plasticity, and its dysregulation contributes to Alzheimer's disease pathology. These findings are in line with a model in which CAP1 controls important aspects during neuron differentiation, while CAP2 is relevant in differentiated neurons. We here report CAP2 expression during neuron differentiation and its enrichment in growth cones. We therefore hypothesized that CAP2 is relevant not only in excitatory synapses, but also in differentiating neurons. However, CAP2 inactivation neither impaired growth cone morphology and motility nor neuron differentiation. Moreover, CAP2 mutant mice did not display any obvious changes in brain anatomy. Hence, differently from CAP1, CAP2 was dispensable for neuron differentiation and brain development. Interestingly, overexpression of CAP2 rescued not only growth cone size in CAP1-deficient neurons, but also their morphology and differentiation. Our data provide evidence for functional redundancy of CAP1 and CAP2 in differentiating neurons, and they suggest compensatory mechanisms in single mutant neurons.

CAP2 contributes to tumorigenesis in gastric cancer by targeting transcription factor SOX9.

BACKGROUND: Gastric cancer (GC) is one of the most common tumors and the major cause of cancer-related mortality in the world. The purpose of this study is to identify new biomarker and reveal its potential molecular mechanism in GC. METHODS: The expression of CAP2 was observed by the bioinformatics analysis and western blot assays. The effects of CAP2 on cell proliferation and growth were tested by MTT assay, EdU assay, colony formation assay, and flow cytometric assay, respectively. ChIP and dual-luciferase assays were confirmed that SOX9 binding sites were putative regulatory elements in the transcriptional activation of CAP2. Furthermore, western blot and xenograft assays were applied to examine whether SOX9 was involved in the regulation of CAP2 expression. RESULTS: We reported that CAP2 is overexpressed in GC cells and tissues and related to a poorer prognosis for GC patients. Moreover, we found that knockdown of CAP2 suppressed the proliferation, growth, and cell cycle of GC cells. Besides, the transcription factor SOX9 participated in the CAP2-mediated proliferation of GC cells in vitro and in vivo. CONCLUSIONS: Our results provide novel evidence that CAP2 plays an essential role in the genesis and development of GC, thus potentially highlighting this gene as a therapeutic target.

Interplay between transcriptional regulators and the SAGA chromatin modifying complex fine-tune iron homeostasis.

The human fungal pathogen Candida albicans responds to iron deprivation by a global transcriptome reconfiguration known to be controlled by the transcriptional regulators Hap43 (also known as Cap2), Sef1, and the trimeric Hap2-Hap3-Hap5 complex. However, the relative roles of these regulators are not known. To dissect this system, we focused on the FRP1 and ACO1 genes, which are induced and repressed, respectively, under iron deprivation conditions. Chromatin immunoprecipitation assays showed that the trimeric HAP complex and Sef1 are recruited to both FRP1 and ACO1 promoters. While the HAP complex occupancy at the FRP1 promoter was Sef1-dependent, occupancy of Sef1 was not dependent on the HAP complex. Furthermore, iron deprivation elicited histone H3-Lys9 hyperacetylation and Pol II recruitment mediated by the trimeric HAP complex and Sef1 at the FRP1 promoter. In contrast, at the ACO1 promoter, the HAP trimeric complex and Hap43 promoted histone deacetylation and also limited Pol II recruitment under iron deprivation conditions. Mutational analysis showed that the SAGA subunits Gcn5, Spt7, and Spt20 are required for C. albicans growth in iron-deficient medium and for H3-K9 acetylation and transcription from the FRP1 promoter. Thus, the trimeric HAP complex promotes FRP1 transcription by stimulating H3K9Ac and Pol II recruitment and, along with Hap43, functions as a repressor of ACO1 by maintaining a deacetylated promoter under iron-deficient conditions. Thus, a regulatory network involving iron-responsive transcriptional regulators and the SAGA histone modifying complex functions as a molecular switch to fine-tune tight control of iron homeostasis gene expression in C. albicans.

Prognostic impact of adenylyl cyclase-associated protein 2 (CAP2) in glioma: A clinicopathological study.

BACKGROUND: Gliomas are a group of diseases arising from intracranial neoplastic tissues that produce a wide spectrum of clinicopathological features and morphological changes. Key questions that intrigue neuro-oncology researchers include defining novel oncophenotypic signatures relevant to diagnosing such tumors and predicting prognoses among patients. One of the key regulators of the cellular actin dynamics is adenylyl cyclase-associated protein 2 (CAP2), a protein that has been studied before in the milieu of cancer and shown to be associated with tumor progression; yet, its expression levels in the context of gliomas have not been assessed. Hence, we were interested in investigating CAP2 expression in gliomas and evaluating its clinicopathological and prognostic significance. MATERIALS AND METHODS: CAP2 expression at the protein level was analyzed in 47 human paraffin-embedded gliomas and normal brain tissues by automated immunohistochemical analysis. Statistical analysis was also performed to assess CAP2 expression level in normal and tumor tissues, and to evaluate its clinicopathological and prognostic significance. RESULTS: Our results revealed high expression of CAP2 protein in tumors of gliomas compared to normal tissues and normal areas adjacent to tumors. High expression of CAP2 was also associated with advanced tumor grades among gliomas. Kaplan-Meier analysis revealed that high CAP2 expression was associated with poor prognosis of patients with glioma (P < 0.05). In Cox regression analysis, CAP2 expression was indicated as an independent prognostic factor for overall survival (hazard ratio (HR) = 1.843, 95% confidence interval (CI), 1.252-2.714; P < 0.005). CONCLUSION: CAP2 is overexpressed in glioma and it is proposed as a potential prognostic biomarker for patients with gliomas. CAP2 expression level may serve as a promising target for diagnosis and treatment of glioma.

A linkage and exome study implicates rare variants of KANK4 and CAP2 in bipolar disorder in a multiplex family.

OBJECTIVES: Bipolar disorder (BD) is a neuropsychiatric disorder with a complex pattern of inheritance. Although many genetic studies have been conducted on BD, its genetic correlates remain uncertain. This study was aimed at  identifying the genetic underpinnings of the disorder in an Indian family, which has been under comprehensive clinical evaluation and follow-up for over 12 years. METHODS: We analysed a four-generation family with several of its members diagnosed for BD employing a combination of genetic linkage and exome analysis. RESULTS: We obtained suggestive LOD score for a chromosome 1 and a chromosome 6 marker (D1S410; LOD = 3.01, Ó¨ = 0; and D6S289; LOD = 1.58, Ó¨ = 0). Manual haplotyping of the regions encompassing these two markers helped delimit a critical genomic interval of 32.44 Mb (D1S2700-D1S435; chromosome 1p31.1-13.2) and another of 10.34 Mb (D6S470-D6S422; chromosome 6p22.3-22.2). We examined the exomic sequences corresponding to these two intervals and found rare variants, NM_181712.4: c.2461G>T (p.Asp821Tyr) in KANK4 at 1p31.1-13.2; and NM_006366:c.-93G>A, in the 5' UTR of CAP2 at 6p22.3-22.2. CONCLUSIONS: Our studysuggests involvement of KANK4 or CAP2 or both in BD in this family. Further analysis of these two genes in BD patients and functional evaluation of the allelic variants identified are suggested.

CAP2 is a Valuable Biomarker for Diagnosis and Prognostic in Patients with Gastric Cancer.

Cyclase-associated protein 2 (CAP2) protein is reported to be upregulated in hepatocellular carcinoma (HCC), human breast cancer, and malignant melanoma. However, its expression in gastric cancer remains unknown, this study was to investigate CAP2 expression and its prognostic significance in gastric cancer. Firstly, we analyzed the Oncomine databases to compare CAP2 mRNA expression in gastric cancer and normal tissues. CAP2 protein expression was analyzed in gastric cancer samples and non-tumor mucosa by RT-PCR and immunohistochemical analysis. Consequently, statistical analyses were performed to evaluate the clinicopathological significance of CAP2 expression in gastric cancer. CAP2 expression was significant higher in gastric cancer tissues than that in non-tumor mucosa at protein levels. CAP2 was up-regulated in 57.8% (252/436) of gastric cancer samples, while detected in only 10.9% (10/92) of non-tumor mucosa. Statistical analysis shows that the expression of CAP2 was correlated with tumor size, Lauren's classification, depth of invasion, lymph node and distant metastases, and regional lymph node stage, TNM stage, but not with age, sex, histology classification, and histologic differentiation. Kaplan-Meier analysis indicated that high CAP2 expression was associated with poor overall survival (78.7%) in 203 of 252 gastic cancer patients. In stage I, II, and III tumors, the 5-year survival rate was lower in those with high expression of CAP2 than those with low expression. In stage IV tumors, the expression of CAP2 did not correlate with the 5-year survival rate. Multiple Cox regression analysis indicated CAP2 as an independent predictor for overall survival [hazard ratio (HR) = 2.045, 95% confidence interval: 1.445-2.895, p < 0.01], while Lauren's classification, TNM stage, and expression of CAP2 were independent prognostic factors in patients with gastric cancer. For the first time, we found that CAP2 was upregulated in gastic cancer, and was associated with lymph node and distant metastases. CAP2 may serve as a prognostic indicator for patients with gastic cancer.

CAP2 mutation leads to impaired actin dynamics and associates with supraventricular tachycardia and dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure and excessive risk of sudden cardiac death. Around half of DCM cases are idiopathic, and genetic factors seem to play an important role. AIM: We investigated a possible genetic cause of DCM in two consanguineous children from a Bedouin family. METHODS AND RESULTS: Using exome sequencing and searching for rare homozygous variations, we identified a nucleotide change in the donor splice consensus sequence of exon 7 in CAP2 as the causative mutation. Using patient-derived fibroblasts, we demonstrated that the mutation causes skipping of exons 6 and 7. The resulting protein is missing 64 amino acids in its N-CAP domain that should prevent its correct folding. CAP2 protein level was markedly reduced without notable compensation by the homolog CAP1. However, ß-actin mRNA was elevated as demonstrated by real-time qPCR. In agreement with the essential role of CAP2 in actin filament polymerization, we demonstrate that the mutation affects the kinetics of repolymerization of actin in patient fibroblasts. CONCLUSIONS: This is the first report of a recessive deleterious mutation in CAP2 and its association with DCM in humans. The clinical phenotype recapitulates the damaging effects on the heart observed in Cap2 knockout mice including DCM and cardiac conduction disease, but not the other effects on growth, viability, wound healing and eye development. Our data underscore the importance of the proper kinetics of actin polymerization for normal function of the human heart.

Multiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation.

Iron is required for growth and metabolism by virtually all organisms. The human fungal pathogen Candida albicans has evolved multiple strategies to acquire iron. The Cap2/Hap43 transcriptional regulator, essential for robust virulence of C. albicans, controls iron homeostasis gene expression by promoter binding and repression of iron utilization genes. The expression of iron uptake genes is also dependent on Cap2, although Cap2 was not recruited to its promoters. Cap2, bearing the conserved bipartite HAP4L-bZIP domain, also contains multiple blocks of amino acids that form the highly conserved carboxyl-terminal region. In this study, we sought to identify the requirements of the different domains for Cap2 function. We constructed a series of mutants bearing either point mutations or deletions in the conserved domains and examined Cap2 activity. Deletion of the highly conserved extreme C-terminal region did not impair expression of Cap2 mutant protein but impaired cell growth and expression of iron homeostasis genes under iron-depleted conditions. Mutations in the amino-terminal HAP4L and basic leucine zipper (bZIP) domains also impaired growth and gene expression. Furthermore, chromatin immunoprecipitation (ChIP) assays showed that the HAP4L domain and the bZIP domain are both essential for Cap2 recruitment to ACO1 and CYC1 promoters. Unexpectedly, the C-terminal conserved region was also essential for Cap2 promoter recruitment. Thus, our results suggest that Cap2 employs multiple evolutionarily conserved domains, including the C-terminal domain for its transcriptional activity.IMPORTANCE Iron is an essential micronutrient for living cells. Candida albicans, the predominant human fungal pathogen, thrives under diverse environments with vastly different iron levels in the mammalian host. Therefore, to tightly control iron homeostasis, C. albicans has evolved a set of transcriptional regulators that cooperate to either upregulate or downregulate transcription of iron uptake genes or iron utilization genes. Cap2/Hap43, a critical transcriptional regulator, contains multiple conserved protein domains. In this study, we carried out mutational analyses to identify the functional roles of the conserved protein domains in Cap2. Our results show that the bZIP, HAP4L, and the C-terminal domain are each required for Cap2 transcriptional activity. Thus, Cap2 employs multiple, disparate protein domains for regulation of iron homeostasis in C. albicans.

[Expression and self-assembly of HIV-1 CAP2NC protein].

We constructed the CAP2NC prokaryotic expression vector of HIV-1 NL4-3 strain and obtained relatively pure CAP2NC protein by optimizing its purification conditions to explore its in vitro self-assembly conditions. Primers were designed according to the CAP2NC DNA sequence of HIV-1 NL4-3 strain. The target gene was amplified by PCR and cloned into prokaryotic expression vector pTO-T7. Then the recombinant strain was transformed into Escherichia coli BL21 (DE3). IPTG induced protein expression, then the protein was purified by hydrophobic chromatography. SDS-PAGE and Western blotting were performed to analyze the target protein, and the biological activity of the antigen was identified through ELISA. The self-assembly of CAP2NC protein was analyzed by transmission electron microscopy and gel filtration chromatography. The protein had good reaction with the specific antibodies of p24 and formed different structures in various conditions. When 10% yeast RNA was added to the protein complex, the recombinant protein only formed into a tubular structure, which was similar to the self-assembled structure of the HIV-1 virus capsid. The results showed that the HIV-1 CAP2NC protein had in vitro self-assembly activity, and the RNA affected the structure of CAP2NC protein assembly. The protein can be used as a simple and effective molecular model to study its structure, and then it can provide a reference for the study of HIV immature virus particles.

Expression and self-assembly of HIV-1 CAP2NC protein / 生物工程学报

We constructed the CAP2NC prokaryotic expression vector of HIV-1 NL4-3 strain and obtained relatively pure CAP2NC protein by optimizing its purification conditions to explore its in vitro self-assembly conditions. Primers were designed according to the CAP2NC DNA sequence of HIV-1 NL4-3 strain. The target gene was amplified by PCR and cloned into prokaryotic expression vector pTO-T7. Then the recombinant strain was transformed into Escherichia coli BL21 (DE3). IPTG induced protein expression, then the protein was purified by hydrophobic chromatography. SDS-PAGE and Western blotting were performed to analyze the target protein, and the biological activity of the antigen was identified through ELISA. The self-assembly of CAP2NC protein was analyzed by transmission electron microscopy and gel filtration chromatography. The protein had good reaction with the specific antibodies of p24 and formed different structures in various conditions. When 10% yeast RNA was added to the protein complex, the recombinant protein only formed into a tubular structure, which was similar to the self-assembled structure of the HIV-1 virus capsid. The results showed that the HIV-1 CAP2NC protein had in vitro self-assembly activity, and the RNA affected the structure of CAP2NC protein assembly. The protein can be used as a simple and effective molecular model to study its structure, and then it can provide a reference for the study of HIV immature virus particles.

Neuronal Actin Dynamics, Spine Density and Neuronal Dendritic Complexity Are Regulated by CAP2.

Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2(gt/gt) mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2(gt/gt) with increased number of excitatory synapses. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2(gt/gt) neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin Ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics.

Overexpression of adenylate cyclase-associated protein 2 is a novel prognostic marker in malignant melanoma.

Malignant melanoma is one of the lethal malignant tumors worldwide. Previously we reported that adenylate cyclase-associated protein 2 (CAP2), which is a well-conserved actin regulator, was overexpressed in hepatocellular carcinoma; however, CAP2 expression in other clinical cancers remains unclear. The aim of the current study was to clarify the clinicopathological significance of CAP2 overexpression in malignant melanoma. Immunohistochemical analyses revealed that many melanoma cells exhibited diffuse cytoplasmic expression of CAP2, whereas no normal melanocytes showed detectable immunostaining for CAP2. A high level of CAP2 expression was seen in 14 of 50 melanomas and was significantly correlated with greater tumor thickness and nodular melanoma subtypes. In addition, a high level of CAP2 expression was associated with poor overall survival in univariate and multivariate analyses. For 13 patients, samples of primary and metastatic melanoma tissue were available: four patients exhibited higher levels of CAP2 expression in metastatic tumor compared to the primary site, whereas no patient showed lower levels of CAP2 expression in metastatic melanomas. Our findings show that CAP2 overexpression is a novel prognostic marker in malignant melanoma and that CAP2 expression seems to increase stepwise during tumor progression, suggesting the involvement of CAP2 in the aggressive behavior of malignant melanoma.

Molecular and functional characterization of the first tick CAP2b (periviscerokinin) receptor from Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The cDNA of the receptor for CAP(2b)/periviscerokinin (PVK) neuropeptides, designated Rhimi-CAP(2b)-R, was cloned from synganglia of tick Rhipicephalus (Boophilus) microplus. This receptor is the ortholog of the insect CAP(2b)/PVK receptor, as concluded from analyses of the predicted protein sequence, phylogenetics and functional expression. Expression analyses of synganglion, salivary gland, Malpighian tubule, and ovary revealed Rhimi-CAP(2b)-R transcripts. The expression in mammalian cells of the open reading frame of Rhimi-CAP(2b)-R cDNA fused with a hemagglutinin tag at the receptor N-terminus was confirmed by immunocytochemistry. In a calcium bioluminescence assay the recombinant receptor was activated by the tick Ixodes scapularis CAP(2b)/PVK and a PVK analog with EC50s of 64 nM and 249 nM, respectively. Tick pyrokinins were not active. This is the first report on the functional characterization of the CAP(2b)/PVK receptor from any tick species which will now permit the discovery of the physiological roles of these neuropeptides in ticks, as neurohormones, neuromodulators and/or neurotransmitters.