LIM-Domain-Binding Protein 1 Mediates Cell Proliferation and Drug Resistance in Colorectal Cancer

Objective: It has been shown that LIM-domain-binding protein 1 (LDB1) is involved in the tumorigenesis of several cancers, but its function in colorectal cancer (CRC) has not been fully explained. This study is aimed to investigate whether LDB1 is involved in regulating the cell growth and drug sensitivity of CRC.Methods: To analyze the protein expression of LDB1 in CRC tissues, western blot was used. KM plotter and UALCAN databases were used to predict the prognosis of CRC patients with low or high LDB1 expression. To do the correlation analysis in CRC tissues, GEPIA database was used. CCK-8 assay and xenograft models were used to evaluate the effects of LDB1 in CRC cell growth. An oxaliplatin-resistant cell line was constructed to evaluate the effect of LDB1 in drug sensitivity of CRC cells.Results: Our current research confirmed that LDB1 was upregulated in CRC tumor tissues, and its elevation predicted a poor prognosis for CRC patients. LDB1 was also found positively correlated with CCNA1, BCL2 and BCLW, but negatively correlated with the pro-apoptotic signals (BID, BAX and BAK). Silence of LDB1 significantly inhibited CRC cell growth in vitro, and CRC cells with low expression of LDB1 had a lower tumorigenesis rate in tumor-bearing nude mice. Our experiments also showed that LDB1 silence enhanced the anti-tumor activity of oxaliplatin in CRC cells. The expression of LDB1 was also found increased in oxaliplatin-resistant CRC cell lines, and silence of LDB1 partly restored the antitumor effect of oxaliplatin in an oxaliplatin-resistant CRC cell line.Conclusion: Our current results revealed the roles of LDB1 in the growth and drug resistance of CRC cells, and may provide the new theoretical support for LDB1 as a potential target for the treatment of CRC in the future.

Structural Aspects of LIMK Regulation and Pharmacology

Malfunction of the actin cytoskeleton is linked to numerous human diseases including neurological disorders and cancer. LIMK1 (LIM domain kinase 1) and its paralogue LIMK2 are two closely related kinases that control actin cytoskeleton dynamics. Consequently, they are potential therapeutic targets for the treatment of such diseases. In the present review, we describe the LIMK conformational space and its dependence on ligand binding. Furthermore, we explain the unique catalytic mechanism of the kinase, shedding light on substrate recognition and how LIMK activity is regulated. The structural features are evaluated for implications on the drug discovery process. Finally, potential future directions for targeting LIMKs pharmacologically, also beyond just inhibiting the kinase domain, are discussed.

The Nature of Diamino Linker and Halogen Bonding Define Selectivity of Pyrrolopyrimidine-Based LIMK1 Inhibitors

The LIM-domain kinase (LIMK) family consists of two isoforms, LIMK1 and LIMK2, which are highly homologous, making selective inhibitor development challenging. LIMK regulates dynamics of the actin cytoskeleton, thereby impacting many cellular functions including cell morphology and motility. Here, we designed and synthesised analogues of a known pyrrolopyrimidine LIMK inhibitor with moderate selectivity for LIMK1 over LIMK2 to gain insights into which features contribute to both activity and selectivity. We incorporated a different stereochemistry around a cyclohexyl central moiety to achieve better selectivity for different LIMK isoforms. Inhibitory activity was assessed by kinase assays, and biological effects in cells were determined using an in vitro wound closure assay. Interestingly, a slight change in stereochemistry alters LIMK isoform selectivity. Finally, a docking study was performed to predict how the new compounds interact with the target.

LIM Domain-Wide Comprehensive Virtual Mutagenesis Provides Structural Rationale for Cardiomyopathy Mutations in CSRP3

Cardiomyopathies are a severe and chronic cardiovascular burden worldwide, affecting a large cohort in the general population. Cysteine and glycine-rich protein 3 (CSRP3) is one of key proteins implicated in dominant dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). In this study, we device a rapid in-silico screening protocol that creates a mutational landscape map for all possible allowed and disallowed substitutions in the protein of interest. This map provides the structural and functional insights on the stability of LIM domains of CSRP3. Further, the sequence analysis delineates the eukaryotic CSRP3 protein orthologs which complements the mutational map. Next, we also evaluated the effect of HCM/DCM mutations on these domains. One of highly destabilising mutations - L44P (also disease causing) and a neutral mutation - L44M were further subjected to molecular dynamics (MD) simulations. The results establish that L44P substitution affects the LIM domain structure. The present study provides a useful perspective to our understanding of the role of mutations in the CSRP3 LIM domains and their evolution. Experimentally verifying every reported mutation can become challenging both in time and resources used. This study provides a novel screening method for quick identification of key mutation sites for specific protein structures that can reduce the burden on experimental research.

Lin11-Isl1-Mec3 Domain Proteins as Mechanotransducers in Endothelial and Vascular Smooth Muscle Cells

Arterial hypertension is the leading risk factor for cardiovascular morbidity and mortality worldwide. However, little is known about the cellular mechanisms underlying it. In small arteries and arterioles, a chronic increase in blood pressure raises wall tension and hence stretches, namely, the medial vascular smooth muscle cells (VSMC) but also endothelial cell (EC) to cell contacts. Initially compensated by an increase in vascular tone, the continuous biomechanical strain causes a prominent change in gene expression in both cell types, frequently driving an arterial inward remodeling process that ultimately results in a reduction in lumen diameter, stiffening of the vessel wall, and fixation of blood pressure, namely, diastolic blood pressure, at the elevated level. Sensing and propagation of this supraphysiological stretch into the nucleus of VSMC and EC therefore seems to be a crucial step in the initiation and advancement of hypertension-induced arterial remodeling. Focal adhesions (FA) represent an important interface between the extracellular matrix and Lin11-Isl1-Mec3 (LIM) domain-containing proteins, which can translocate from the FA into the nucleus where they affect gene expression. The varying biomechanical cues to which vascular cells are exposed can thus be rapidly and specifically propagated to the nucleus. Zyxin was the first protein described with such mechanotransducing properties. It comprises 3 C-terminal LIM domains, a leucine-rich nuclear export signal, and N-terminal features that support its association with the actin cytoskeleton. In the cytoplasm, zyxin promotes actin assembly and organization as well as cell motility. In EC, zyxin acts as a transcription factor, whereas in VSMC, it has a less direct effect on mechanosensitive gene expression. In terms of homology and structural features, lipoma preferred partner is the nearest relative of zyxin among the LIM domain proteins. It is almost exclusively expressed by smooth muscle cells in the adult, resides like zyxin at FA but seems to affect mechanosensitive gene expression indirectly, possibly via altering cortical actin dynamics. Here, we highlight what is currently known about the role of these LIM domain proteins in mechanosensing and transduction in vascular cells.

Mutations in CSRP3/MLP, a Z-disc associated gene are functionally associated with dilated cardiomyopathy in Indian population

CSRP3 is a LIM domain containing protein, known to play an important role in cardiomyocyte development, differentiation and pathology. Mutations in CSRP3 gene are reported in both dilated and hypertrophic cardiomyopathy (DCM and HCM), however, the genotype-phenotype correlation still remains elusive. To investigate the pathogenic potential of CSRP3 variants in our DCM cohort, we have screened 100 DCM cases and 100 controls and identified 3 non-synonymous variations, of which two are missense variants viz., c.233 GGC>GTC, p.G78V; c.420 TGG>TGC, p.W140C, and the third one is a single nucleotide polymorphism (SNP) c.46 ACC>TCC, p.T16S. These variants were absent from 100 control individuals (200 chromosomes). In vitro functional analysis has revealed reduction of CSRP3 protein level in stably-transfected C2C12 cells with p.G78V or p.W140C variants. Immunostaining demonstrates both cytoplasmic and nuclear localization of the wild-type protein, however variants p.G78V and p.W140C cause obvious reduction in the cytoplasmic expression of CSRP3 protein which is more pronounced in case of p.W140C. Disarrayed actin cytoskeleton was also observed in mutants. Besides, the expression of target genes namely Ldb3, Myoz2, Tcap, Tnni3 and Ttn are also downregulated in response to these variants. GST-pulldown assay has also showed a diminished binding of CSRP3 protein with α-Actinin due to both variants p.G78V and p.W140C. Both 2D, 3D-modeling have shown confirmational changes. Most in silico tools predict these variants as deleterious. Taken together, all these results suggest the impaired gene function due to these deleterious variants in CSRP3, implicating its possible disease causing role in DCM.

TRIP6 functions in brain ciliogenesis

TRIP6, a member of the ZYXIN-family of LIM domain proteins, is a focal adhesion component. Trip6 deletion in the mouse, reported here, reveals a function in the brain: ependymal and choroid plexus epithelial cells are carrying, unexpectedly, fewer and shorter cilia, are poorly differentiated, and the mice develop hydrocephalus. TRIP6 carries numerous protein interaction domains and its functions require homodimerization. Indeed, TRIP6 disruption in vitro (in a choroid plexus epithelial cell line), via RNAi or inhibition of its homodimerization, confirms its function in ciliogenesis. Using super-resolution microscopy, we demonstrate TRIP6 localization at the pericentriolar material and along the ciliary axoneme. The requirement for homodimerization which doubles its interaction sites, its punctate localization along the axoneme, and its co-localization with other cilia components suggest a scaffold/co-transporter function for TRIP6 in cilia. Thus, this work uncovers an essential role of a LIM-domain protein assembly factor in mammalian ciliogenesis.

A Novel Circular RNA circITSN2 Targets the miR-218-5p/LMO7 Axis to Promote Chicken Embryonic Myoblast Proliferation and Differentiation

Circular RNA (circRNA) is a class of endogenous non-coding RNAs without 5′ and 3′ ends; an increasing number of studies show that circRNA is involved in skeletal muscle development. From our previous sequencing data, the circRNAome in breast muscle of two chicken lines with a distinct rate of muscle development, which included a fast muscle growing broiler (FMGB) and a slow muscle growing layer (SMGL), we found a novel differentially expressed circRNA generated by intersectin 2 (ITSN2) gene (named circITSN2). We verified that circITSN2 is a skeletal muscle-enriched circRNA that promotes chicken primary myoblast (CPM) proliferation and differentiation. Further molecular mechanism analysis of circITSN2 in chicken myogenesis was performed, and we found circITSN2 directly targeting miR-218-5p. Besides, miR-218-5p inhibits CPM proliferation and differentiation, which is contrary to circITSN2. Commonly, circRNAs act as a miRNA sponge to alleviate the inhibition of miRNAs on mRNAs. Thus, we also identified that a downstream gene LIM domain 7 (LMO7) was inhibited by miR-218-5p, while circITSN2 could block the inhibitory effect of miR-218-5p by targeting it. Functional analysis revealed that LMO7 also accelerates CPM proliferation and differentiation, which was similar to circITSN2 but contrary to miR-218-5p. Taken together, these results suggested that circITSN2 promotes chicken embryonic skeletal muscle development via relieving the inhibition of miR-218-5p on LMO7. Our findings revealed a novel circITSN2/miR-218-5p/LMO7 axis in chicken embryonic skeletal muscle development, which expands our understanding of the complex muscle development regulatory network.