Structural domain in the Titin N2B-us region binds to FHL2 in a force-activation dependent manner.

Titin N2B unique sequence (N2B-us) is a 572 amino acid sequence that acts as an elastic spring to regulate muscle passive elasticity. It is thought to lack stable tertiary structures and is a force-bearing region that is regulated by mechanical stretching. In this study, the conformation of N2B-us and its interaction with four-and-a-half LIM domain protein 2 (FHL2) are investigated using AlphaFold2 predictions and single-molecule experimental validation. Surprisingly, a stable alpha/beta structural domain is predicted and confirmed in N2B-us that can be mechanically unfolded at forces of a few piconewtons. Additionally, more than twenty FHL2 LIM domain binding sites are predicted to spread throughout N2B-us. Single-molecule manipulation experiments reveals the force-dependent binding of FHL2 to the N2B-us structural domain. These findings provide insights into the mechano-sensing functions of N2B-us and its interactions with FHL2.

Genome-wide analysis of the mouse LIM gene family reveals its roles in regulating pathological cardiac hypertrophy.

LIM-domain proteins have been shown to be associated with heart development and diseases. Systematic studies of LIM family members at the genome-wide level, which are crucial to further understand their functions in cardiac hypertrophy, are currently lacking. Here, 70 LIM genes were identified and characterised in mice. The expression patterns of LIM genes differ greatly during cardiac development and in the case of hypertrophy. Both Crip2 and Xirp2 are differentially expressed in cardiac hypertrophy and during heart failure. In addition, the hypertrophic state of cardiomyocytes is controlled by the relative expression levels of Crip2 and Xirp2. This study provides a foundation for further understanding of the special roles of LIM proteins in mammalian cardiac development and hypertrophy.

Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer.

BACKGROUND: Despite recent advances in diagnostic and therapeutic approaches for gastric cancer (GC), the survival of patients with advanced GC remains very low. Islet-1 (ISL1) is a LIM-homeodomain transcription factor, which is upregulated and promotes cell proliferation in GC. The exact mechanism by which ISL1 influences GC development is unclear. METHODS: Co-immunoprecipitation (co-IP) and glutathione S-transferase (GST)-pulldown assays were employed to evaluate the interaction of ISL1 with CDK1. Western blot and immunohistochemistry analyses were performed to evaluate the ability of CDK1 to phosphorylate ISL1 at Ser 269 in GC cell and tissue specimens. Chromatin immunoprecipitation (ChIP), ChIP re-IP, luciferase reporter, and CCK-8 assays were combined with flow cytometry cell cycle analysis to detect the transactivation potency of ISL1-S269-p and its ability to promote cell proliferation. The self-stability and interaction with CDK1 of ISL1-S269-p were also determined. RESULTS: ISL1 is phosphorylated by CDK1 at serine 269 (S269) in vivo. Phosphorylation of ISL1 by CDK1 on serine 269 strengthened its binding on the cyclin B1 and cyclin B2 promoters and increased its transcriptional activity in GC. Furthermore, CDK1-dependent phosphorylation of ISL1 correlated positively with ISL1 protein self-stability in NIH3T3 cells. CONCLUSIONS: ISL1-S269-p increased ISL1 transcriptional activity and self-stability while binding to the cyclinB1 and cyclinB2 promoters promotes cell proliferation. ISL1-S269-p is therefore crucial for tumorigenesis and potentially a direct therapeutic target for GC.

Structural and Functional Characterization of Host FHL1 Protein Interaction with Hypervariable Domain of Chikungunya Virus nsP3 Protein.

Decades of insufficient control have resulted in unprecedented spread of chikungunya virus (CHIKV) around the globe, and millions have suffered from the highly debilitating disease. Nevertheless, the current understanding of CHIKV-host interactions and adaptability of the virus to replication in mosquitoes and mammalian hosts is still elusive. Our new study shows that four-and-a-half LIM domain protein (FHL1) is one of the host factors that interact with the hypervariable domain (HVD) of CHIKV nsP3. Unlike G3BPs, FHL1 is not a prerequisite of CHIKV replication, and many commonly used cell lines do not express FHL1. However, its expression has a detectable stimulatory effect(s) on CHIKV replication, and Fhl1 knockout (KO) cell lines demonstrate slower infection spread. Nuclear magnetic resonance (NMR)-based studies revealed that the binding site of FHL1 in CHIKV nsP3 HVD overlaps that of another proviral host factor, CD2AP. The structural data also demonstrated that FHL1-HVD interaction is mostly determined by the LIM1 domain of FHL1. However, it does not mirror binding of the entire protein, suggesting that other LIM domains are involved. In agreement with previously published data, our biological experiments showed that interactions of CHIKV HVD with CD2AP and FHL1 have additive effects on the efficiency of CHIKV replication. This study shows that CHIKV mutants with extensive modifications of FHL1- or both FHL1- and CD2AP-binding sites remain viable and develop spreading infection in multiple cell types. Our study also demonstrated that other members of the FHL family can bind to CHIKV HVD and thus may be involved in viral replication.IMPORTANCE Replication of chikungunya virus (CHIKV) is determined by a wide range of host factors. Previously, we have demonstrated that the hypervariable domain (HVD) of CHIKV nsP3 contains linear motifs that recruit defined families of host proteins into formation of functional viral replication complexes. Now, using NMR-based structural and biological approaches, we have characterized the binding site of the cellular FHL1 protein in CHIKV HVD and defined the biological significance of this interaction. In contrast to previously described binding of G3BP to CHIKV HVD, the FHL1-HVD interaction was found to not be a prerequisite of viral replication. However, the presence of FHL1 has a stimulatory effect on CHIKV infectivity and, subsequently, the infection spread. FHL1 and CD2AP proteins were found to have overlapping binding sites in CHIKV HVD and additive proviral functions. Elimination of the FHL1-binding site in the nsP3 HVD can be used for the development of stable, attenuated vaccine candidates.

Mutation in a flexible linker modulates binding affinity for modular complexes.

Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which ß-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4-ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.

Heterozygous LHX3 mutations may lead to a mild phenotype of combined pituitary hormone deficiency.

LHX3 is an LIM domain transcription factor involved in the early steps of pituitary ontogenesis. We report here functional studies of three allelic variants, including the first heterozygous variant of LHX3 NM_178138.5(LHX3):c.587T>C (p.(Leu196Pro)) that may be responsible for a milder phenotype of hypopituitarism. Our functional studies showed that NM_178138.5(LHX3):c.587T>C (p.(Leu196Pro)) was not able to activate target promoters in vitro, as it did not bind DNA, and likely affected LHX3 function via a mechanism of haplo-insufficiency. Our study demonstrates the possibility that patients with a heterozygous variant of LHX3 may have pituitary deficiencies, with a milder phenotype than patients with homozygous variants. It is thus of vital to propose an optimal follow-up of such patients, who, until now, were considered as not being at risk of presenting pituitary deficiency. The second variant NM_178138.5(LHX3):c.622C>G (p.(Arg208Gly)), present in a homozygous state, displayed decreased transactivating ability without loss of binding capacity in vitro, concordant with in silico analysis; it should thus be considered to affect LHX3 function. In contrast, the NM_178138.5(LHX3):c.929G>C (p.(Arg310Pro)) variant, in a heterozygous state, also predicted as deleterious in silico, proved functionally active in vitro, and should thus still be classified as a variant of unknown significance. Our study emphasizes the need for functional studies due to the limits of software-based predictions of new variants, and the possible association of a pituitary phenotype to heterozygous LHX3 variants.

Could the interaction between LMX1B and PAX2 influence the severity of renal symptoms?

Nail Patella syndrome (NPS) is a rare autosomal dominant disease characterized by varying degrees of patella, nail, and elbows dysplasia and also ocular and renal congenital abnormalities. The renal involvement, ranging from hematuria and proteinuria to end-stage renal disease, is present in 22-60% of NPS cases. Heterozygous variants in LMX1B are known to be responsible of NPS and it has been hypothesized that the variable expressivity is due to the interaction of LMX1B with other developmental genes. We reported a case of co-presence of LMX1B and PAX2 variants in a child with extrarenal manifestation of NPS and end-stage renal disease but congenital bilateral renal hypodysplasia and vesicoureteral reflux. The LMX1B variant was de novo, whereas the PAX2 variant was inherited from the mother that had bilateral renal hypoplasia although in presence of only a mild chronic kidney disease. The molecular interaction between LMX1B and PAX2 has been already reported in vitro and this finding suggest that the worst renal NPS phenotype of our patient could be due to the defective expression of these two genes during nephrogenesis. In conclusion, our finding suggests that PAX2 may act as modifier gene in Nail Patella phenotype.

A variant in LMX1A causes autosomal recessive severe-to-profound hearing impairment.

Hereditary hearing impairment is a common sensory disorder that is genetically and phenotypically heterogeneous. In this study, we used a homozygosity mapping and exome sequencing strategy to study a consanguineous Pakistani family with autosomal recessive severe-to-profound hearing impairment. This led to the identification of a missense variant (p.Ile369Thr) in the LMX1A gene affecting a conserved residue in the C-terminus of the protein, which was predicted damaging by an in silico bioinformatics analysis. The p.Ile369Thr variant disrupts several C-terminal and homeodomain residue interactions, including an interaction with homeodomain residue p.Val241 that was previously found to be involved in autosomal dominant progressive HI. LIM-homeodomain factor Lmx1a is expressed in the inner ear through development, shows a progressive restriction to non-sensory epithelia, and is important in the separation of the sensory and non-sensory domains in the inner ear. Homozygous Lmx1a mutant mice (Dreher) are deaf with dysmorphic ears with an abnormal morphogenesis and fused and misshapen sensory organs; however, computed tomography performed on a hearing-impaired family member did not reveal any cochleovestibular malformations. Our results suggest that LMX1A is involved in both human autosomal recessive and dominant sensorineural hearing impairment.

Disparate binding kinetics by an intrinsically disordered domain enables temporal regulation of transcriptional complex formation.

Intrinsically disordered regions are highly represented among mammalian transcription factors, where they often contribute to the formation of multiprotein complexes that regulate gene expression. An example of this occurs with LIM-homeodomain (LIM-HD) proteins in the developing spinal cord. The LIM-HD protein LHX3 and the LIM-HD cofactor LDB1 form a binary complex that gives rise to interneurons, whereas in adjacent cell populations, LHX3 and LDB1 form a rearranged ternary complex with the LIM-HD protein ISL1, resulting in motor neurons. The protein-protein interactions within these complexes are mediated by ordered LIM domains in the LIM-HD proteins and intrinsically disordered LIM interaction domains (LIDs) in LDB1 and ISL1; however, little is known about how the strength or rates of binding contribute to complex assemblies. We have measured the interactions of LIM:LID complexes using FRET-based protein-protein interaction studies and EMSAs and used these data to model population distributions of complexes. The protein-protein interactions within the ternary complexes are much weaker than those in the binary complex, yet surprisingly slow LDB1:ISL1 dissociation kinetics and a substantial increase in DNA binding affinity promote formation of the ternary complex over the binary complex in motor neurons. We have used mutational and protein engineering approaches to show that allostery and modular binding by tandem LIM domains contribute to the LDB1LID binding kinetics. The data indicate that a single intrinsically disordered region can achieve highly disparate binding kinetics, which may provide a mechanism to regulate the timing of transcriptional complex assembly.

Interactions between LHX3- and ISL1-family LIM-homeodomain transcription factors are conserved in Caenorhabditis elegans.

LIM-Homeodomain (LIM-HD) transcription factors are highly conserved in animals where they are thought to act in a transcriptional 'LIM code' that specifies cell types, particularly in the central nervous system. In chick and mammals the interaction between two LIM-HD proteins, LHX3 and Islet1 (ISL1), is essential for the development of motor neurons. Using yeast two-hybrid analysis we showed that the Caenorhabditis elegans orthologs of LHX3 and ISL1, CEH-14 and LIM-7 can physically interact. Structural characterisation of a complex comprising the LIM domains from CEH-14 and a LIM-interaction domain from LIM-7 showed that these nematode proteins assemble to form a structure that closely resembles that of their vertebrate counterparts. However, mutagenic analysis across the interface indicates some differences in the mechanisms of binding. We also demonstrate, using fluorescent reporter constructs, that the two C. elegans proteins are co-expressed in a small subset of neurons. These data show that the propensity for LHX3 and Islet proteins to interact is conserved from C. elegans to mammals, raising the possibility that orthologous cell specific LIM-HD-containing transcription factor complexes play similar roles in the development of neuronal cells across diverse species.

The Enterococcus faecalis virulence factor ElrA interacts with the human Four-and-a-Half LIM Domains Protein 2.

The commensal bacterium Enterococcus faecalis is a common cause of nosocomial infections worldwide. The increasing prevalence of multi-antibiotic resistant E. faecalis strains reinforces this public health concern. Despite numerous studies highlighting several pathology-related genetic traits, the molecular mechanisms of E. faecalis virulence remain poorly understood. In this work, we studied 23 bacterial proteins that could be considered as virulence factors or involved in the Enterococcus interaction with the host. We systematically tested their interactions with human proteins using the Human ORFeome library, a set of 12,212 human ORFs, in yeast. Among the thousands of tested interactions, one involving the E. faecalis virulence factor ElrA and the human protein FHL2 was evidenced by yeast two-hybrid and biochemically confirmed. Further molecular characterizations allowed defining an FHL2-interacting domain (FID) of ElrA. Deletion of the FID led to an attenuated in vivo phenotype of the mutated strain clearly indicating that this interaction is likely to contribute to the multifactorial virulence of this opportunistic pathogen. Altogether, our results show that FHL2 is the first host cellular protein directly targeted by an E. faecalis virulence factor and that this interaction is involved in Enterococcus pathogenicity.

Lhx8 interacts with a novel germ cell-specific nuclear factor containing an Nbl1 domain in rainbow trout (Oncorhynchus mykiss).

Lhx8 is an important transcription factor that is preferentially expressed in germ cells. Lhx8 null mice are infertile due to lack of oocytes and impairment of the transition from primordial follicles to primary follicles. Lhx8 deficiency also affects the expression of many important oocyte-specific genes. In this study, we report the characterization of rainbow trout lhx8 genes and identification of a novel germ cell-specific nuclear factor that interacts with Lhx8. Two lhx8 genes, lhx8a and lhx8b, were identified, encoding proteins of 344 and 361 amino acids, respectively. The two proteins share 83% sequence identity and both transcripts are specifically expressed in the ovary. Quantitative real time PCR analysis demonstrated that both genes are expressed highly in pre-vitellogenic ovaries as well as in early stage embryos. Using a yeast two-hybrid screening system, a novel protein (Borealin-2) interacting with Lhx8 was identified. The interaction between either Lhx8a or Lhx8b and Borealin-2 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Borealin-2 is a protein of 255 amino acids containing an Nbl1 domain, and its mRNA expression is restricted to the ovary and testis. A GFP reporter assay revealed that Borealin-2 is a nuclear protein. Collectively, results indicate that both Lhx8a and Lhx8b function through interaction with Borealin-2, which may play an important role during oogenesis and early embryogenesis in rainbow trout.

Protein-protein interactions of the LIM-only protein FHL2 and functional implication of the interactions relevant in cardiovascular disease.

FHL2 belongs to the LIM-domain only proteins and contains four and a half LIM domains, each of which are composed of two zinc finger structures. FHL2 exhibits specific interaction with proteins exhibiting diverse functions, including transmembrane receptors, transcription factors and transcription co-regulators, enzymes, and structural proteins. The function of these proteins is regulated by FHL2, which modulates intracellular signal transduction pathways involved in a plethora of cellular tasks. The present review summarizes the current knowledge on the protein interactome of FHL2 and provides an overview of the functional implication of these interactions in apoptosis, migration, and regulation of nuclear receptor function. FHL2 was originally identified in the heart and there is extensive literature available on the role of FHL2 in the cardiovascular system, which is also summarized in this review.

Critical Roles of the LIM Domains of Lhx3 in Recruiting Coactivators to the Motor Neuron-Specifying Isl1-Lhx3 Complex.

During spinal cord development, the LIM domains of the LIM homeodomain factor Lhx3 bind to either the LIM cofactor nuclear LIM interactor (NLI) or another LIM homeodomain factor, Isl1, assembling the tetrameric V2 interneuron-specifying Lhx3 complex (2NLI:2Lhx3) or the hexameric motor neuron-specifying Isl1-Lhx3 complex (2NLI:2Isl1:2Lhx3). However, the detailed molecular basis by which the Lhx3-LIM domains contribute to motor neuron specification still remains poorly understood. Here, we show that the Lhx3-LIM domains are essential for recruiting transcriptional coactivators to the Isl1-Lhx3 complex. Using a yeast genetic screening system, we identify Lhx3 point mutants that bind to NLI but not Isl1. Accordingly, these mutants fail to assemble the Isl1-Lhx3 complex. However, their interaction with coactivators is relatively intact, and they are fully functional in the Lhx3 complex and V2 interneuron specification. Interestingly, when these Lhx3 mutants are directly fused to Isl1, their transcriptional activity in the Isl1-Lhx3 complex is restored. We further show that this restoration reflects an unexpected role of the Lhx3-LIM domains, likely together with Isl1, to form an interaction interface for coactivators. Our results suggest that the Lhx3-LIM domains play critical roles in transactivation of the Isl1-Lhx3 complex by not only directing the assembly of the Isl1-Lhx3 complex but also recruiting coactivators to the complex.

Muscle LIM Protein: Master regulator of cardiac and skeletal muscle functions.

Muscle LIM Protein (MLP) has emerged as a key regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3 (CSRP3), the gene encoding MLP, are causative of human cardiomyopathies, whereas altered expression patterns are observed in human failing heart and skeletal myopathies. In vitro and in vivo evidences reveal a complex and diverse functional role of MLP in striated muscle, which is determined by its multiple interacting partners and subcellular distribution. Experimental evidence suggests that MLP is implicated in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum of its intracellular roles still unfolds.

Novel Lethal Form of Congenital Hypopituitarism Associated With the First Recessive LHX4 Mutation.

BACKGROUND: LHX4 encodes a member of the LIM-homeodomain family of transcription factors that is required for normal development of the pituitary gland. To date, only incompletely penetrant heterozygous mutations in LHX4 have been described in patients with variable combined pituitary hormone deficiencies. OBJECTIVE/HYPOTHESIS: To report a unique family with a novel recessive variant in LHX4 associated with a lethal form of congenital hypopituitarism that was identified through screening a total of 97 patients. METHOD: We screened 97 unrelated patients with combined pituitary hormone deficiency, including 65% with an ectopic posterior pituitary, for variants in the LHX4 gene using Sanger sequencing. Control databases (1000 Genomes, dbSNP, Exome Variant Server, ExAC Browser) were consulted upon identification of variants. RESULTS: We identified the first novel homozygous missense variant (c.377C>T, p.T126M) in two deceased male patients of Pakistani origin with severe panhypopituitarism associated with anterior pituitary aplasia and posterior pituitary ectopia. Both were born small for gestational age with a small phallus, undescended testes, and mid-facial hypoplasia. The parents' first-born child was a female with mid-facial hypoplasia (DNA was unavailable). Despite rapid commencement of hydrocortisone and T4 in the brothers, all three children died within the first week of life. The LHX4(p.T126M) variant is located within the LIM2 domain, in a highly conserved location. The absence of homozygosity for the variant in over 65 000 controls suggests that it is likely to be responsible for the phenotype. CONCLUSION: We report, for the first time to our knowledge, a novel homozygous mutation in LHX4 associated with a lethal phenotype, implying that recessive mutations in LHX4 may be incompatible with life.

Alternative splicing of the LIM-homeodomain transcription factor Isl1 in the mouse retina.

Islet-1 (Isl1) is a LIM-homeodomain (LIM-HD) transcription factor that functions in a combinatorial manner with other LIM-HD proteins to direct the differentiation of distinct cell types within the central nervous system and many other tissues. A study of pancreatic cell lines showed that Isl1 is alternatively spliced generating a second isoform, Isl1ß, which is missing 23 amino acids within the C-terminal region. This study examines the expression of the canonical and alternative Isl1 transcripts across other tissues, in particular, within the retina, where Isl1 is required for the differentiation of multiple neuronal cell types. The alternative splicing of Isl1 is shown to occur in multiple tissues, but the relative abundance of Isl1α and Isl1ß expression varies greatly across them. In most tissues, Isl1α is the more abundant transcript, but in others the transcripts are expressed equally, or the alternative splice variant is dominant. Within the retina, differential expression of the two Isl1 transcripts increases as a function of development, with dynamic changes in expression peaking at E16.5 and again at P10. At the cellular level, individual retinal ganglion cells vary in their expression, with a subset of small-to-medium sized cells expressing only the alternative isoform. The functional significance of the difference in protein sequence between the two Isl1 isoforms was also assessed using a luciferase assay, demonstrating that the alternative isoform forms a less effective transcriptional complex for activating gene expression. These results demonstrate the differential presence of the canonical and alternative isoforms of Isl1 amongst retinal ganglion cell classes. As Isl1 participates in the differentiation of multiple cell types within the CNS, the present results support a role for alternative splicing in the establishment of cellular diversity in the developing nervous system.

LIM proteins in actin cytoskeleton mechanoresponse.

The actin cytoskeleton assembles into branched networks or bundles to generate mechanical force for critical cellular processes such as establishment of polarity, adhesion, and migration. Stress fibers (SFs) are contractile actomyosin structures that physically couple to the extracellular matrix through integrin-based focal adhesions (FAs), thereby transmitting force into and across the cell. Recently, LIN-11, Isl1, and MEC-3 (LIM) domain proteins have been implicated in mediating this cytoskeletal mechanotransduction. Among the more well-studied LIM domain adapter proteins is zyxin, a dynamic component of both FAs and SFs. Here we discuss recent research detailing the mechanisms by which SFs adjust their structure and composition to balance mechanical forces and suggest ways that zyxin and other LIM domain proteins mediate mechanoresponse.

Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes.

PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1 (four-and-a-half LIM domains protein 1) and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs (neonatal rat ventricular myocytes) respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP-PKD1 fusion protein expressed in NRVMs. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α1-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.