Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins.
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ABSTRACT:
SUBMITTER: Siddiqui MQ
PROVIDER: S-EPMC7961639 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Previous studies have demonstrated that the adhesion-plaque protein, zyxin, interacts specifically with a 23 kDa protein, called the cysteine-rich protein 1 (CRP1), which has been implicated in myogenesis. Primary sequence analyses have revealed that both zyxin and CRP1 exhibit multiple copies of a structural motif called the LIM domain. LIM domains, which are defined by the consensus CX2CX16-23HX2CX2CX2CX16-23CX2-3(C,H,D), are found in a variety of proteins that are involved in cell growth and differentiation. Recent studies have established that LIM domains are zinc-binding structures that mediate specific protein-protein interactions. For example, in the case of the zyxin-CRP1 interaction, one of zyxin's three LIM domains is necessary and sufficient for binding to CRP1. Because the CRP1 molecule is comprised primarily of two LIM domains, we were interested in the possibility that the binding site for zyxin on CRP1 might also be contained within a single LIM domain. Consistent with the hypothesis that the LIM domains of CRP1 are critical for the protein's zyxin-binding function, zinc-depleted CRP1 displays a reduced zyxin-binding activity. However, domain mapping analyses have revealed that neither of the two individual LIM domains of CRP1 can support a wild-type interaction with zyxin. Collectively, our results suggest that the binding site for zyxin on CRP1 is not contained within a single contiguous sequence of amino acids. Instead, the interaction appears to rely on the co-ordinate action of a number of residues that are displayed in both of CRP1's LIM domains.
| S-EPMC1219431 | biostudies-other
Project description:Contractile actomyosin stress fibers are critical for maintaining the force balance between the interior of the cell and its environment. Consequently, the actin cytoskeleton undergoes dynamic mechanical loading. This results in spontaneous, stochastic, highly localized strain events, characterized by thinning and elongation within a discrete region of stress fiber. Previous work showed the LIM-domain adaptor protein, zyxin, is essential for repair and stabilization of these sites. Using live imaging, we show paxillin, another LIM-domain adaptor protein, is also recruited to stress fiber strain sites. Paxillin recruitment to stress fiber strain sites precedes zyxin recruitment. Zyxin and paxillin are each recruited independently of the other. In cells lacking paxillin, actin recovery is abrogated, resulting in slowed actin recovery and increased incidence of catastrophic stress fiber breaks. For both paxillin and zyxin, the LIM domains are necessary and sufficient for recruitment. This work provides further evidence of the critical role of LIM-domain proteins in responding to mechanical stress in the actin cytoskeleton.
| S-EPMC3749209 | biostudies-literature
Project description:Interaction with extracellular matrix can trigger a variety of responses by cells including changes in specific gene expression and cell differentiation. The mechanism by which cell surface events are coupled to the transcriptional machinery is not understood, however, proteins localized at sites of cell-substratum contact are likely to function as signal transducers. We have recently purified and characterized a low abundance adhesion plaque protein called zyxin (Crawford, A. W., and M. C. Beckerle. 1991. J. Biol. Chem. 266:5847-5853; Crawford, A. W., J. W. Michelsen, and M. C. Beckerle. 1992. J. Cell Biol. 116:1381-1393). We have now isolated and sequenced zyxin cDNA and we report here that zyxin exhibits an unusual proline-rich NH2-terminus followed by three tandemly arrayed LIM domains. LIM domains have previously been identified in proteins that play important roles in transcriptional regulation and cellular differentiation. LIM domains have been proposed to coordinate metal ions and we have demonstrated by atomic absorption spectroscopy that purified zyxin binds zinc, a result consistent with the idea that zyxin has zinc fingers. In addition, we have discovered that zyxin interacts in vitro with a 23-kD protein that also exhibits LIM domains. Microsequence analysis has revealed that the 23-kD protein (or cCRP) is the chicken homologue of the human cysteine-rich protein (hCRP). By double-label indirect immunofluorescence, we found that zyxin and cCRP are extensively colocalized in chicken embryo fibroblasts, consistent with the idea that they interact in vivo. We conclude that LIM domains are zinc-binding sequences that may be involved in protein-protein interactions. The demonstration that two cytoskeletal proteins, zyxin and cCRP, share a sequence motif with proteins important for transcriptional regulation raises the possibility that zyxin and cCRP are components of a signal transduction pathway that mediates adhesion-stimulated changes in gene expression.
| S-EPMC2289750 | biostudies-other
Project description:Neuronal dendrites have specialized actin-rich structures called dendritic spines that receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic morphology and stability are largely mediated by regulation of the actin cytoskeleton; however, the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important role in the postsynaptic development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 are enriched in dendritic spines, and their knockdown impairs spine development and synapse formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine stabilization. Importantly, the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stability and dendritic arbor complexity. These findings identify LASP1 and LASP2 as novel regulators of neuronal circuitry.SIGNIFICANCE STATEMENT Proper regulation of the actin cytoskeleton is essential for the structural stability of dendrites and dendritic spines. Consequently, the malformation of dendritic structures accompanies numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are best known for their role in regulating the stabilization and function of actin thin filaments in muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly expressed in the brain, little is currently known about their function in the nervous system. In this study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation and long-term maintenance of dendrites and dendritic spines.
| S-EPMC6961999 | biostudies-literature
Project description:Cellular responses to mechanical perturbation are vital to cell physiology. In particular, migrating cells have been shown to sense substrate stiffness and alter cell morphology and speed. Zyxin is a focal adhesion protein that responds to external mechanical forces; however, the mechanisms of zyxin recruitment at force-bearing sites are unknown. Using force-sensing microfabricated substrates, we simultaneously measured traction force and zyxin recruitment at force-bearing sites. GFP-tagged zyxin accumulates at force-bearing sites at the leading edge, but not at the trailing edge, of migrating epithelial cells. Zyxin recruitment at force-bearing sites depends on Rho-kinase and myosin II activation, suggesting that zyxin responds not only to the externally applied force, as previously shown, but also to the internally generated actin-myosin force. Zyxin in turn recruits vasodilator-stimulated phosphoprotein, a regulator of actin assembly, to force-bearing sites. To dissect the domains of zyxin that are essential for this unique force-dependent accumulation, we generated two zyxin truncation mutants: one lacking the LIM domain (?LIM) and one containing only the LIM domain with all three LIM motifs (LIM). GFP-tagged ?LIM does not localize to the force-bearing sites, but GFP-tagged zyxin LIM-domain is sufficient for the recruitment to and dynamics at force-bearing focal adhesions. Furthermore, one or two LIM motifs are not sufficient for force-dependent accumulation, suggesting that all three LIM motifs are required. Therefore, the LIM domain of zyxin recruits zyxin to force-bearing sites at the leading edge of migrating cells.
| S-EPMC3164133 | biostudies-literature
Project description:Zyxin is a dual-function LIM domain protein that regulates actin dynamics in response to mechanical stress and shuttles between focal adhesions and the cell nucleus. Here we show that zyxin contributes to UV-induced apoptosis. Exposure of wild-type fibroblasts to UV-C irradiation results in apoptotic cell death, whereas cells harboring a homozygous disruption of the zyxin gene display a statistically significant survival advantage. To gain insight into the molecular mechanism by which zyxin promotes apoptotic signaling, we expressed an affinity-tagged zyxin variant in zyxin-null cells and isolated zyxin-associated proteins from cell lysates under physiological conditions. A 130-kDa protein that was co-isolated with zyxin was identified by microsequence analysis as the Cell Cycle and Apoptosis Regulator Protein-1 (CARP-1). CARP-1 associates with the LIM region of zyxin. Zyxin lacking the CARP-1 binding region shows reduced proapoptotic activity in response to UV-C irradiation. We demonstrate that CARP-1 is a nuclear protein. Zyxin is modified by phosphorylation in cells exposed to UV-C irradiation, and nuclear accumulation of zyxin is induced by UV-C exposure. These findings highlight a novel mechanism for modulating the apoptotic response to UV irradiation.
| S-EPMC2941191 | biostudies-literature
Project description:Chromobox (CBX) family proteins control chromatin structure and gene expression. However, the functions of CBXs in cancer progression, especially breast cancer, are inadequately studied. We assessed the significance of eight CBX proteins in breast cancer. We performed immunohistochemistry and bioinformatic analysis of data from Oncomine, GEPIA Dataset, bcGenExMiner, Kaplan-Meier Plotter, and cBioPortal. We compared mRNA and protein expression levels of eight CBX proteins between breast tumor and normal tissue. The expression difference of CBX7 was the greatest, and CBX7 was downregulated in breast cancer tissues compared with normal breast tissues. The expression of CBX2 was strongly associated with tumor stage. We further analyzed the association between the eight CBX proteins and the following clinicopathological features: menopause age, estrogen receptor (ER), progesterone receptor (PR) and HER-2 receptor status, nodal status, P53 status, triple-negative status, and the Scarff-Bloom-Richardson grade (SBR) and Nottingham prognostic index (NPI). Survival analysis in the Kaplan-Meier Plotter database showed that the eight CBX proteins were significantly associated with prognosis. Moreover, CBX genes in breast cancer patients had a high net alteration frequency of 57%. There were significant co-expression correlations between the following CBX protein pairs: CBX4 positively with CBX8, CBX6 positively with CBX7, and CBX2 negatively with CBX7. We also analyzed the Gene Ontology enrichment of the CBX proteins, including biological processes, cellular components, and molecular functions. CBX 1/2/3/5/8 may be oncogenes for breast cancer, whereas CBX 6 and 7 may be tumor suppressors for breast cancer. All eight CBX proteins may be predictive for prognosis. Clinical trials are needed to confirm the significance of the eight CBX proteins in breast cancer.
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Project description:The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.
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Project description:The CCN proteins are key signalling and regulatory molecules involved in many vital biological functions, including cell proliferation, angiogenesis, tumourigenesis and wound healing. How these proteins influence such a range of functions remains incompletely understood but is probably related to their discrete modular nature and a complex array of intra- and inter-molecular interactions with a variety of regulatory proteins and ligands. Although certain aspects of their biology can be attributed to the four individual modules that constitute the CCN proteins, recent results suggest that some of their biological functions require cooperation between modules. Indeed, the modular structure of CCN proteins provides important insight into their structure-function relationships.
| S-EPMC2683937 | biostudies-literature
Project description:BackgroundPectins are plant cell wall polysaccharides that can be acetylated on C2 and/or C3 of galacturonic acid residues. The degree of acetylation of pectin can be modulated by pectin acetylesterase (EC 3.1.1.6, PAE). The function and structure of plant PAEs remain poorly understood and the role of the fine-tuning of pectin acetylation on cell wall properties has not yet been elucidated.ResultsIn the present study, a bioinformatic approach was used on 72 plant PAEs from 16 species among 611 plant PAEs available in plant genomic databases. An overview of plant PAE proteins, particularly Arabidopsis thaliana PAEs, based on phylogeny analysis, protein motif identification and modeled 3D structure is presented. A phylogenetic tree analysis using protein sequences clustered the plant PAEs into five clades. AtPAEs clustered in four clades in the plant kingdom PAE tree while they formed three clades when a phylogenetic tree was performed only on Arabidopsis proteins, due to isoform AtPAE9. Primitive plants that display a smaller number of PAEs clustered into two clades, while in higher plants, the presence of multiple members of PAE genes indicated a diversification of AtPAEs. 3D homology modeling of AtPAE8 from clade 2 with a human Notum protein showed an α/β hydrolase structure with the hallmark Ser-His-Asp of the active site. A 3D model of AtPAE4 from clade 1 and AtPAE10 from clade 3 showed a similar shape suggesting that the diversification of AtPAEs is unlikely to arise from the shape of the protein. Primary structure prediction analysis of AtPAEs showed a specific motif characteristic of each clade and identified one major group of AtPAEs with a signal peptide and one group without a signal peptide. A multiple sequence alignment of the putative plant PAEs revealed consensus sequences with important putative catalytic residues: Ser, Asp, His and a pectin binding site. Data mining of gene expression profiles of AtPAE revealed that genes from clade 2 including AtPAE7, AtPAE8 and AtPAE11, which are duplicated genes, are highly expressed during plant growth and development while AtPAEs without a signal peptide, including AtPAE2 and AtPAE4, are more regulated in response to plant environmental conditions.ConclusionBioinformatic analysis of plant, and particularly Arabidopsis, AtPAEs provides novel insights, including new motifs that could play a role in pectin binding and catalytic sites. The diversification of AtPAEs is likely to be related to neofunctionalization of some AtPAE genes.
| S-EPMC5465549 | biostudies-literature