SH3 Domain Protein Research Articles

The emergence of nebulin repeats and evolution of lasp family proteins.

The LIM and SH3 domain protein (lasp) family, the smallest proteins in the nebulin superfamily, consists of vertebrate lasp-1 expressed in various non-muscle tissues, vertebrate lasp-2 expressed in the brain and cardiac muscle, and invertebrate lasp whose functions have been analyzed in Ascidiacea and Insecta. Gene evolution of the lasp family proteins was investigated by multiple alignments, comparison of gene structure, and synteny analyses in eukaryotes in which mRNA expression was confirmed. All invertebrates analyzed in this study belonging to the clade Filasterea, with the exception of Placozoa, have at least one lasp gene. The minimal actin-binding region (LIM domain and first nebulin repeat) and SH3 domain detected in vertebrate lasp-2 were found to be conserved among the lasp family proteins, and we showed that nematode lasp has actin-binding activity. The linker sequences vary among invertebrate lasp proteins, implying that the lasp family proteins have universal and diverse functions. Gene structures and syntenic analyses suggest that a gene fragment encoding two nebulin repeats and a linker emerged in Filasterea or Holozoa, and the first lasp gene was generated following combination of three gene fragments encoding the LIM domain, two nebulin repeats with a linker, and the SH3 domain.

Fold Evolution before LUCA: Common Ancestry of SH3 Domains and OB Domains.

SH3 and OB are the simplest, oldest, and most common protein domains within the translation system. SH3 and OB domains are β-barrels that are structurally similar but are topologically distinct. To transform an OB domain to a SH3 domain, β-strands must be permuted in a multistep and evolutionarily implausible mechanism. Here, we explored relationships between SH3 and OB domains of ribosomal proteins, initiation, and elongation factors using a combined sequence- and structure-based approach. We detect a common core of SH3 and OB domains, as a region of significant structure and sequence similarity. The common core contains four β-strands and a loop, but omits the fifth β-strand, which is variable and is absent from some OB and SH3 domain proteins. The structure of the common core immediately suggests a simple permutation mechanism for interconversion between SH3 and OB domains, which appear to share an ancestor. The OB domain was formed by duplication and adaptation of the SH3 domain core, or vice versa, in a simple and probable transformation. By employing the folding algorithm AlphaFold2, we demonstrated that an ancestral reconstruction of a permuted SH3 sequence folds into an OB structure, and an ancestral reconstruction of a permuted OB sequence folds into a SH3 structure. The tandem SH3 and OB domains in the universal ribosomal protein uL2 share a common ancestor, suggesting that the divergence of these two domains occurred before the last universal common ancestor.

Long non‑coding RNA H19 regulates LASP1 expression in osteosarcoma by competitively binding to miR‑29a‑3p.

A prevalent type of bone tumor, osteosarcoma (OS) is prone to pulmonary metastasis, which results in a high relapse risk and poor prognosis for patients. The progression of OS is significantly associated with the expression of long non-coding (lnc)RNA H19. To the best of our knowledge, however, the exact molecular mechanism of this lncRNA has not been fully investigated. The present study verified the effect of H19 on the proliferation and invasion of osteosarcoma cells via in vivo and in vitro experiments, including Cell Counting Kit-8, western blot, reverse transcription-quantitative PCR, wound healing and Transwell assays. H19 was found to be overexpressed in OS compared with corresponding normal adjacent tissue. In addition, H19 served as a competing endogenous ncRNA targeting microRNA-29a-3p and activating LIM and SH3 domain protein 1 and modulating the OS cell phenotype. The results of the present study may improve understanding of OS pathogenesis.

The role of Bag3 in cell signaling.

Bag3 has been implicated in a wide variety of physiological processes from autophagy to aggresome formation and from cell transformation to survival. We argue that involvement of Bag3 in many of these processes is due to its distinct function in cell signaling. The structure of Bag3 suggests that it can serve as a scaffold that links molecular chaperones Hsp70 and small Hsps with components of a variety of signaling pathways. Major protein-protein interaction motifs of Bag3 that recruit components of signaling pathways are WW domain and PXXP motif that interacts with SH3-domain proteins. Furthermore, Hsp70-Bag3 appears to be a sensor of abnormal polypeptides during the proteotoxic stress. It also serves as a sensor of a mechanical force during mechanotransduction. Common feature of these and probably certain other sensory mechanisms is that they represent responses to specific kinds of abnormal proteins, i.e. unfolded filamin A in case of mechanotransduction or stalled translating polypeptides in case of sensing proteasome inhibition. Overall Hsp70-Bag3 module represents a novel signaling node that responds to multiple stimuli and controls multiple physiological processes.

HOTTIP Enhances Gemcitabine and Cisplatin Resistance Through Sponging miR-637 in Cholangiocarcinoma.

Chemo-resistance prominently hampers the effects of systemic chemotherapy to cholangiocarcinoma (CCA). Long non-coding RNAs (lncRNAs) have been shown to have great importance not only in tumorigenesis but also in therapeutic prognosis. The aim of this study is to investigate the role of lncRNA HOTTIP in the chemo-resistance to cisplatin and gemcitabine (CG) in CCA. The upregulated expression of HOTTIP was observed in CCA patients and the upregulation was associated with therapeutic responsiveness and prognosis. HOTTIP silencing powerfully increased the chemotherapy sensitivity through weakening proliferation and colony formation and increasing apoptosis. Subsequently, miR-637 was identified as the functional target of HOTTIP, since mechanically it could be targeted by HOTTIP and functionally its overexpression dismissed the changes by HOTTIP silencing in vitro and in vivo. Moreover, LIM and SH3 domain protein 1 (LASP1) could be targeted and regulated by miR-637. In all, HOTTIP modulates the sensitivity to CG in CCA through the HOTTIP/miR-637/LASP1 regulatory axis, providing a new opportunities for CCA treatment.

Abstract 2450: A systemic approach to decipher the interactome of RET receptor isoforms

Abstract The REarranged during Transfection (RET) receptor tyrosine kinase is pivotal for normal tissue development, but is also an oncogene driver involved in several human cancers. Alternative splicing at the 3' end of the RET gene leads to expression of two conserved protein isoforms, RET9 and RET51, that differ in their subcellular localization and protein trafficking, as well as their functional roles in tumorigenesis and metastatic processes. Importantly, RET9 and RET51 have unique C-terminal phospho-tyrosine binding sites, suggesting that they may also differ in their interactomes. We used a combination of cell-based screening and in silico approaches to identify novel potential interaction partners of RET isoforms. We performed a Mammalian Membrane Two-Hybrid (MaMTH) screen using a library of SH2 domain-containing adaptor and signaling proteins, to identify interactions with each RET isoform. We complemented these studies by using sequence homology detection models (HMM, PSSM), based on SH2 domain sequences known to interact with RET, to rank the SH2 library members and predict novel interactions. Independently, we compared published consensus binding sequences for each SH2 domain library member with predicted RET phosphotyrosine motifs to identify potential interactors. Predicted interactions were validated in co-immunoprecipitation assays. We confirmed previously known interactions of RET with SH2 domain proteins including SHC1, GRB2 and GRB10, and identified additional novel RET-binding proteins, a subset of which showed differential interactions that were mediated through RET isoform-specific docking sites. Our results suggest that combinations of distinct interaction partners may contribute to RET isoform-specific functions. Together, our research has developed a systematic approach to map and characterize RET isoform interactions. Our data suggest that no single method identified all confirmed RET interactions, and that a combination of multiple approaches improves characterization of growth factor receptor interactomes. Citation Format: Samira Kheitan, Annika E. Pedersen, Brandy D. Hyndman, Luka Drecun, Punit Saraon, Igor Stagliar, Lois M. Mulligan. A systemic approach to decipher the interactome of RET receptor isoforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2450.

The effects of testosterone on gene expression of cell-adhesion molecules and scaffolding proteins: The role of sex in early development.

Sex-specific differences in brain plasticity appear to be organised by testosterone, which is particularly important during the early stages of development. The main purpose of the present study was to examine the sex differences in mRNA and protein levels of selected cell-adhesion molecules and scaffolding proteins on postnatal days 5 (P5) and 9 (P9) in the rat hippocampus, as well as evaluate the effects of testosterone treatment (100nM, 48hr) on synaptic proteins in SH-SY5Y (neuron-like) and U-87MG (astrocyte-like) cells. The gene expression levels of Neuroligin 3 and 'SH3 and multiple ankyrin repeat domains protein' 1 and 3 (SHANK1 and SHANK3) were significantly lower in males compared to females at P5. At P9, a similar significant trend towards a decrease in mRNA expression and protein levels of SHANK3 was found in males. Testosterone treatment induced a significant decrease of Neuroligin 1-3 mRNA expression in both SH-SY5Y and U-87MG cells. SHANK1 and SHANK3 mRNA levels significantly decreased in U-87MG cells response to testosterone presence. The presented results demonstrate that the association of selected postsynaptic cell-adhesion molecules and scaffolding proteins is sex-related. Testosterone appears to be particularly involved in the developmental mechanisms related to neuroplasticity.

Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/β-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.

Distinctive alteration of presynaptic proteins in the outer molecular layer of the dentate gyrus in Alzheimer's disease.

Synaptic degeneration has been reported as one of the best pathological correlates of cognitive deficits in Alzheimer’s disease. However, the location of these synaptic alterations within hippocampal sub-regions, the vulnerability of the presynaptic versus postsynaptic compartments, and the biological mechanisms for these impairments remain unknown. Here, we performed immunofluorescence labelling of different synaptic proteins in fixed and paraffin-embedded human hippocampal sections and report reduced levels of several presynaptic proteins of the neurotransmitter release machinery (complexin-1, syntaxin-1A, synaptotagmin-1 and synaptogyrin-1) in Alzheimer’s disease cases. The deficit was restricted to the outer molecular layer of the dentate gyrus, whereas other hippocampal sub-fields were preserved. Interestingly, standard markers of postsynaptic densities (SH3 and multiple ankyrin repeat domains protein 2) and dendrites (microtubule-associated protein 2) were unaltered, as well as the relative number of granule cells in the dentate gyrus, indicating that the deficit is preferentially presynaptic. Notably, staining for the axonal components, myelin basic protein, SMI-312 and Tau, was unaffected, suggesting that the local presynaptic impairment does not result from axonal loss or alterations of structural proteins of axons. There was no correlation between the reduction in presynaptic proteins in the outer molecular layer and the extent of the amyloid load or of the dystrophic neurites expressing phosphorylated forms of Tau. Altogether, this study highlights the distinctive vulnerability of the outer molecular layer of the dentate gyrus and supports the notion of presynaptic failure in Alzheimer’s disease.

Circ_0023028 contributes to the progression of laryngeal squamous cell carcinoma by upregulating LASP1 through miR-486-3p.

Circular RNAs (circRNAs) are implicated in the tumorigenesis of human cancers. However, the effects of circRNAs on laryngeal squamous cell carcinoma (LSCC) are largely unknown. Here, we aimed to explore the roles of circ_0023028 in LSCC development. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure circ_0023028, miR-486-3p, and Lin-Isl-Mec (LIM) and SH3 domain protein 1 (LASP1) mRNA. The characteristics of circ_0023028 were determined by RNase R digestion assay and Actinomycin D assay. Cell Counting Kit-8 (CCK-8) assay and colony formation assay were utilized for cell proliferation. Transwell assay was adopted for cell invasion and migration. Flow cytometry analysis was carried out to analyze cell cycle and apoptosis. RNA pull-down assay and dual-luciferase reporter assay were used to explore the association between miR-486-3p and circ_0023028 or LASP1. Western blot assay was adopted to measure LASP1 protein level. Murine xenograft model was executed to investigate the function of circ_0023028 in vivo. High expression of circ_0023028 was observed in LSCC tissues and cells. Circ_0023028 was stable and possessed a loop structure. Circ_0023028 silencing suppressed LSCC cell proliferation, metastasis and cell cycle process and induced apoptosis in vitro and hampered tumor growth in vivo. Further mechanism analysis demonstrated that circ_0023028 could sponge miR-486-3p to regulate LASP1 expression in LSCC cells. The malignant behaviors of LSCC cells mediated by circ_0023028 knockdown were rescued by the inhibition of miR-486-3p. Moreover, miR-486-3p suppressed LSCC cell progression via binding to LASP1. Circ_0023028 knockdown might impede the progression of LSCC by regulating miR-486-3p/LASP1 axis, which could provide a novel insight on the mechanism of LSCC progression.

Hepatitis B virus X protein promotes vimentin expression via LIM and SH3 domain protein 1 to facilitate epithelial-mesenchymal transition and hepatocarcinogenesis

BackgroundHepatitis B virus (HBV) X protein (HBX) has been reported to be responsible for the epithelial-mesenchymal transition (EMT) in HBV-related hepatocellular carcinoma (HCC). Vimentin is an EMT-related molecular marker. However, the importance of vimentin in the pathogenesis of HCC mediated by HBX has not been well determined.MethodsThe expression of vimentin induced by HBX, and the role of LIM and SH3 domain protein 1 (LASP1) in HBX-induced vimentin expression in hepatoma cells were examined by western blot and immunohistochemistry analysis. Both the signal pathways involved in the expression of vimentin, the interaction of HBX with vimentin and LASP1, and the stability of vimentin mediated by LASP1 in HBX-positive cells were assessed by western blot, Co-immunoprecipitation, and GST-pull down assay. The role of vimentin in EMT, proliferation, and migration of HCC cells mediated by HBX and LASP1 were explored with western blot, CCK-8 assay, plate clone formation assay, transwell assay, and wound healing assay.ResultsVimentin expression was increased in both HBX-positive hepatoma cells and HBV-related HCC tissues, and the expression of vimentin was correlated with HBX in HBV-related HCC tissues. Functionally, vimentin was contributed to the EMT, proliferation, and migration of hepatoma cells mediated by HBX. The mechanistic analysis suggested that HBX was able to enhance the expression of vimentin through LASP1. On the one hand, PI3-K, ERK, and STAT3 signal pathways were involved in the upregulation of vimentin mediated by LASP1 in HBX-positive hepatoma cells. On the other hand, HBX could directly interact with vimentin and LASP1, and dependent on LASP1, HBX was capable of promoting the stability of vimentin via protecting it from ubiquitination mediated protein degradation. Besides these, vimentin was involved in the growth and migration of hepatoma cells mediated by LASP1 in HBX-positive hepatoma cells.ConclusionTaken together, these findings demonstrate that, dependent on LASP1, vimentin is crucial for HBX-mediated EMT and hepatocarcinogenesis, and may serve as a potential target for HBV-related HCC treatment.34DcRyoit96E3KKn7g7GiGVideo abstract

Multiple Rare Risk Coding Variants in Postsynaptic Density-Related Genes Associated With Schizophrenia Susceptibility.

ObjectiveSchizophrenia is a chronic debilitating neurobiological disorder of aberrant synaptic connectivity and synaptogenesis. Postsynaptic density (PSD)–related proteins in N-methyl-D-aspartate receptor–postsynaptic signaling complexes are crucial to regulating the synaptic transmission and functions of various synaptic receptors. This study examined the role of PSD-related genes in susceptibility to schizophrenia.MethodsWe resequenced 18 genes encoding the disks large-associated protein (DLGAP), HOMER, neuroligin (NLGN), neurexin, and SH3 and multiple ankyrin repeat domains (SHANK) protein families in 98 schizophrenic patients with family psychiatric history using semiconductor sequencing. We analyzed the protein function of the identified rare schizophrenia-associated mutants via immunoblotting and immunocytochemistry.ResultsWe identified 50 missense heterozygous mutations in 98 schizophrenic patients with family psychiatric history, and in silico analysis revealed some as damaging or pathological to the protein function. Ten missense mutations were absent from the dbSNP database, the gnomAD (non-neuro) dataset, and 1,517 healthy controls from Taiwan BioBank. Immunoblotting revealed eight missense mutants with altered protein expressions in cultured cells compared with the wild type.ConclusionOur findings suggest that PSD-related genes, especially the NLGN, SHANK, and DLGAP families, harbor rare functional mutations that might alter protein expression in some patients with schizophrenia, supporting contributing rare coding variants into the genetic architecture of schizophrenia.

Hippocampal LASP1 ameliorates chronic stress-mediated behavioral responses in a mouse model of unpredictable chronic mild stress

Substantial evidence has revealed that abnormalities in synaptic plasticity play important roles during the process of depression. LASP1 (LIM and SH3 domain protein 1), a member of actin-binding proteins, has been shown to be associated with the regulation of synaptic plasticity. However, the role of LASP1 in the regulation of mood is still unclear. Here, using an unpredictable chronic mild stress (UCMS) paradigm, we found that the mRNA and protein levels of LASP1 were decreased in the hippocampus of stressed mice and that UCMS-induced down-regulation of LASP1 was abolished by chronic administration of fluoxetine. Adenosine-associated virus-mediated hippocampal LASP1 overexpression alleviated the UCMS-induced behavioral results of forced swimming test and sucrose preference test in stressed mice. It also restored the dendritic spine density, elevated the levels of AKT (a serine/threonine protein kinase), phosphorylated-AKT, insulin-like growth factor 2, and postsynaptic density protein 95. These findings suggest that LASP1 alleviates UCMS-provoked behavioral defects, which may be mediated by an enhanced dendritic spine density and more activated AKT-dependent LASP1 signaling, pointing to the antidepressant role of LASP1.

LncRNA NEAT1 Knockdown Inhibits Retinoblastoma Progression by miR-3619-5p/LASP1 Axis.

Retinoblastoma (RB) is the most common intraocular tumor in childhood. Long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NTAT1) has been reported to be related to RB progression. This study aims to study the molecular mechanism of NEAT1 in regulating cell cycle, proliferation, apoptosis, migration, and invasion in RB. The expression levels of NEAT1 and miR-3619-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of LIM and SH3 domain protein 1 (LASP1) was measured by western blot. The proliferation of RB cells was analyzed by cell counting kit-8 (CCK-8) and cell colony formation assays. Cell migration and invasion were evaluated by transwell assay. Cell cycle and apoptosis were assessed by flow cytometry analysis. The association between miR-3619-5p and NEAT1 or LASP1 was predicted by starBase 3.0 database and identified by dual-luciferase reporter assay. The effects of NEAT1 knockdown on the tumor growth in vivo were detected by in vivo tumor formation assay. NEAT1 expression was dramatically up-regulated, and miR-3619-5p expression was obviously downregulated in RB tissues and cells compared with control groups. The protein level of LASP1 was obviously increased in RB tissues or cells relative to paracancerous normal tissues or cells, respectively. Functionally, NEAT1 silencing inhibited RB cell migration, invasion, and proliferation, whereas induced cell apoptosis and cell cycle arrest in RB; this phenomenon was partially abolished by miR-3619-5p inhibitor. Mechanistically, NEAT1 acted as a sponge of miR-3619-5p, and miR-3619-5p was associated with LASP1. In addition, NEAT1 knockdown decreased the volume and weight of RB tumor in vivo. Together, NEAT1 silencing repressed cell migration, invasion, and proliferation, whereas induced cell apoptosis and cycle arrest by sponging miR-3619-5p to inhibit LASP1 expression in RB cells. This study may provide a theoretical basis for RB therapy.

Up-regulated microRNA-218-5p ameliorates the damage of dopaminergic neurons in rats with Parkinson’s disease via suppression of LASP1

ObjectiveParkinson’s disease (PD) is a frequent degenerative disease of the nervous system with undefined pathogenesis. This study explored the protective effect of microRNA (miR)-218−5p on dopaminergic neuron injury in substantia nigra (SN) of rats with PD through the regulation of LIM and SH3 domain protein 1 (LASP1). MethodsThe PD rat model was established by fixed point injection of 6-hydroxydopamine into the rats. The PD rats were injected with miR-218−5p overexpressed recombinant adeno-associated virus (rAAV) or LASP1 silenced rAAV to explore their roles in dopaminergic neurons in SN of rats with PD. The changes in pathological structure of SN were observed and the expression of tyrosine hydroxylase (TH) and deacetylvindoline acetyltransferase (DAT), the dopaminergic neuron apoptosis and oxidative stress factor in the SN were detected. The expression of miR-218−5p, LASP1, Bcl-2 and Bax in SN was detected. The targeting relationship between miR-218−5p and LASP1 was confirmed. ResultsDeclined miR-218−5p and overexpressed LASP1 existed in the brain SN of PD rats. Up-regulated miR-218−5p or inhibited LASP1 improved the pathological damage of dopaminergic neurons and increased the number of TH and DAT positive cells in brain SN of PD rats. Furthermore, elevated miR-218−5p or depressed LASP1 inhibited the apoptosis, and oxidative stress of dopaminergic neurons in brain SN of PD rats. In addition, increased miR-218−5p repressed the expression of LASP1 in the brain SN of PD rats. LASP1 was proven to be a direct target of miR-218−5p. ConclusionThe study highlights that up-regulated miR-218−5p could improve the damage of dopaminergic neurons in PD rats, which was related to the inhibition of LASP1.

LINC01503/miR-342-3p facilitates malignancy in non-small-cell lung cancer cells via regulating LASP1

BackgroundNon-small cell lung cancer (NSCLC) is one of the major types of lung cancer, which is a prevalent human disease all over the world. LncRNA LINC01503 is a super-enhancer-driven long non-coding RNA that is dysregulated in several types of human cancer. However, its role in NSCLC remains unknown.MethodsThirty NSCLC patients were recruited between April 2012 and April 2016. Luciferase reporter assay, qRT-PCR, Cell Counting Kit-8 (CCK-8), Transwell migration assay, RNA pull-down assay, western blotting, 5-ethynyl-29-deoxyuridine (EdU) assays, and flow cytometry were utilized to characterize the roles and relationships among LINC01503, miR-342-3p, and LASP1 in NSCLC. The transplanted mouse model was built to examine their biological functions in vivo.ResultsWe demonstrated that the expression of lncRNA LINC01503 and LIM and SH3 domain protein 1 (LASP1) were upregulated and miR-342-3p was downregulated in NSCLC samples and cell lines. Functional experiments revealed that inhibiting the expression of LINC01503 or over-expression of miR-342-3p inhibited NSCLC growth and metastasis both in vitro and in vivo. In addition, LINC01503 could bind to miR-342-3p and affect the expression of LASP1.ConclusionThese results provide a comprehensive analysis of the roles of LINC01503 as a competing endogenous RNA (ceRNA) in NSCLC progression.

Nephrotoxicity Profile of Cadmium Revealed by Proteomics in Mouse Kidney.

Cadmium (Cd) is a highly toxic metal and kidney is its main target. However, the molecular effects and associated potential impacts of Cd-accumulated kidney have not been well investigated. In this study, mouse was used as a model to investigate the Cd-induced proteomic profile change in kidney, and a total of 34 differentially expressed proteins were detected by two-dimensional gel electrophoresis (2-DE) and further identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Through Gene Ontology analysis and KEGG pathway annotation, it showed that Cd-regulated kidney metabolism and promoted renal damage and cell migration. By validation of Western blotting and RT-qPCR, metastasis-related proteins LIM and SH3 domain protein 1 (LASP1) and phosphoenolpyruvate carboxykinase/cytosolic [GTP] (PEPCK1) were confirmed to be upregulated; Acyl-CoA synthetase medium-chain family member 3 (ACSM3) was downregulated. Furthermore, carcinoma development-related proteins initiation factor 4A (eIF4A) and pyridoxine-5'-phosphate oxidase (PNPO) were upregulated, and pyridoxal kinase (PK) was downregulated. The downregulation of Na(+)/H(+) exchange regulatory cofactor (NHERF3) might promote renal damage which associated with decrease of transferrin (TRF) in kidney. Taken together, our results revealed proteomic profile of Cd-induced nephrotoxicity and provided data for further insights into the mechanisms of Cd toxicity.

Dynamic Change of Shanks Gene mRNA Expression and DNA Methylation in Epileptic Rat Model and Human Patients.

Synaptic protein shanks (SH3 and multiple ankyrin repeat domains protein, Shank) have emerged as an important mediator of synaptic remodeling. Synaptic remodeling is a common pathogenic process in various neurological disorders including epilepsy. However, the expression and function of shanks gene in epileptogenesis has not been investigated to date. Herein, we investigated the expression of shanks (shank1/2/3) mRNA expression in both epileptic rats and epilepsy patients. Furthermore, methyl target sequencing was applied to explore the relationship between shank mRNA expression and DNA methylation in both rats and human patients. In general rat model, shank1/2/3 mRNA was downregulated at acute stage, upregulated at latent stage, and returned to the basal level at chronic stage. Ten CpG sites of shank1 was found differentially methylated, out of which 6 were hypermethylated. Seventeen CpG sites of shank3 were differentially methylated, out of which 8 were hypermethylated. In human epilepsy patients, decreased shank2 mRNA was detected from the brain tissue, with DNA hypermethylation dominant from both brain (18 out of 30) and blood tissue (58 out of 80), indicating the regulation role of DNA methylation on shank2 expression. In conclusion, our finding suggests the participation of the shanks gene in the pathophysiology of seizure, out of which 2 shank3 CpG sites (Chr7: 130473419, and Chr7: 130473405) may play an important role in shank3 expression at both the acute and latent stages in the SE rat model.

The roles of SHANK1 in the development of colon cancer.

SH3 and multiple ankyrin repeat domains protein 1 (SHANK1) belongs to a family of postsynaptic scaffolding proteins. In this study, we found that SHANK1 was abnormally high expressed in colon cancer tissues compared to normal tissues. Colon cancer patients with low SHANK1 expression had better prognosis. Furthermore, the expression of SHANK1 was knocked down in human colon cancer cell lines HCT116 and HT29 and the role of SHANK1 was investigated in colon cancer tumorigenesis. Our results showed that the knockdown of SHANK1 inhibited the survival and proliferation of both cells. The migration of these two cell lines was significantly reduced and the apoptosis was induced compared with control cells. The Bax/Bcl-2 ratio in both cell lines that SHANK1 was knocked down was increased, which is a signal that the mitochondrial apoptotic pathway was triggered. In addition, we observed that knockdown of SHANK1 reduced the expression of phosphorylated forms of AKT and mTOR. These data suggested that loss of SHANK1 inhibited viability and induced apoptosis of HCT116 and HT29 cells through the AKT/mTOR signaling pathway. Our data revealed that SHANK1 played important roles in the growth of colon cancer cells and may be used as a novel strategy for colon cancer therapy. SIGNIFICANCE OF THE STUDY: Herein, we reported that SHANK1 was abnormally high expressed in colon cancer tissues and associated with worse prognosis of patients. In addition, knockdown of SHANK1 inhibited viability and induced apoptosis in colon cancer cell lines through AKT/mTOR signaling pathways. These data suggest that SHANK1 may be a new oncogene in colon cancer. This study reveals the role of SHANK1 in addition to neuronal development and cognitive development. And it provides a new potential target for the prediction and treatment of colon cancer.

Nonstructural NS5A Protein Regulates LIM and SH3 Domain Protein 1 to Promote Hepatitis C Virus Propagation.

Hepatitis C virus (HCV) propagation is highly dependent on cellular proteins. To identify the host factors involved in HCV propagation, we previously performed protein microarray assays and identified the LIM and SH3 domain protein 1 (LASP-1) as an HCV NS5A-interacting partner. LASP-1 plays an important role in the regulation of cell proliferation, migration, and protein-protein interactions. Alteration of LASP-1 expression has been implicated in hepatocellular carcinoma. However, the functional involvement of LASP1 in HCV propagation and HCV-induced pathogenesis has not been elucidated. Here, we first verified the protein interaction of NS5A and LASP-1 by both in vitro pulldown and coimmunoprecipitation assays. We further showed that NS5A and LASP-1 were colocalized in the cytoplasm of HCV infected cells. NS5A interacted with LASP-1 through the proline motif in domain I of NS5A and the tryptophan residue in the SH3 domain of LASP-1. Knockdown of LASP-1 increased HCV replication in both HCV-infected cells and HCV subgenomic replicon cells. LASP-1 negatively regulated viral propagation and thereby overexpression of LASP-1 decreased HCV replication. Moreover, HCV propagation was decreased by wild-type LASP-1 but not by an NS5A binding-defective mutant of LASP-1. We further demonstrated that LASP-1 was involved in the replication stage of the HCV life cycle. Importantly, LASP-1 expression levels were increased in persistently infected cells with HCV. These data suggest that HCV modulates LASP-1 via NS5A in order to regulate virion levels and maintain a persistent infection.