Spectrin Repeats Research Articles

Alpha-actinin of the chlorarchiniophyte Bigelowiella natans.

The genome of the chlorarchiniophyte Bigelowiella natans codes for a protein annotated as an α-actinin-like protein. Analysis of the primary sequence indicate that this protein has the same domain structure as other α-actinins, a N-terminal actin-binding domain and a C-terminal calmodulin-like domain. These two domains are connected by a short rod domain, albeit long enough to form a single spectrin repeat. To analyse the functional properties of this protein, the full-length protein as well as the separate domains were cloned and isolated. Characerisation showed that the protein is capable of cross-linking actin filaments into dense bundles, probably due to dimer formation. Similar to human α-actinin, calcium-binding occurs to the most N-terminal EF-hand motif in the calmodulin-like C-terminal domain. The results indicate that this Bigelowiella protein is a proper α-actinin, with all common characteristics of a typical α-actinin.

The spectrum of manifestations in desmoplakin gene (DSP) spectrin repeat 6 domain mutations: Immunophenotyping and response to ustekinumab

The immune abnormalities underlying the ichthyoses are poorly understood. To determine the immunophenotype of an ichthyosis resulting from mutations in the spectrin repeat 6 (SR6) domain of desmoplakin gene (DSP) and target therapy on the basis of molecular pathogenesis. Immunophenotyping was performed by using the blood and skin of a girl with SR6 region DSP mutations causing erythroderma/ichthyosis and cardiomyopathy. On the basis of the discovery of T helper 1 and T helper 17/interleukin 23 skewing in the skin and T helper 17/interleukin 22 skewing in blood, ustekinumab therapy was initiated. Ustekinumab was also administered to a boy with an SR6 region DSP mutation and ichthyosis without cardiomyopathy. Both children responded despite previous poor responses to immunosuppressants and retinoids. Small number of patients and immunophenotyping in only 1 patient. An understanding of the molecular basis of inflammation in rare cutaneous disorders can lead to targeted therapy, which promises to be more beneficial than broad immunosuppressants.

The mechano-sensing role of the unique SH3 insertion in plakin domains revealed by Molecular Dynamics simulations

The plakin family of proteins, important actors in cross-linking force-bearing structures in the cell, contain a curious SH3 domain insertion in their chain of spectrin repeats (SRs). While SH3 domains are known to mediate protein-protein interactions, here, its canonical binding site is autoinhibited by the preceding SR. Under force, however, this SH3 domain could be released, and possibly launch a signaling cascade. We performed large-scale force-probe molecular dynamics simulations, across two orders of magnitude of loading rates, to test this hypothesis, on two prominent members of the plakin family: desmoplakin and plectin, obligate proteins at desmosomes and hemidesmosomes, respectively. Our simulations show that force unravels the SRs and abolishes the autoinhibition of the SH3 domain, an event well separated from the unfolding of this domain. The SH3 domain is free and fully functional for a significant portion of the unfolding trajectories. The rupture forces required for the two proteins significantly decrease when the SH3 domain is removed, which implies that the SH3 domain also stabilizes this junction. Our results persist across all simulations, and support a force-sensing as well as a stabilizing role of the unique SH3 insertion, putting forward this protein family as a new class of mechano-sensors.

Delineating SPTAN1 associated phenotypes: from isolated epilepsy to encephalopathy with progressive brain atrophy.

De novo in-frame deletions and duplications in the SPTAN1 gene, encoding the non-erythrocyte αII spectrin, have been associated with severe West syndrome with hypomyelination and pontocerebellar atrophy. We aimed at comprehensively delineating the phenotypic spectrum associated with SPTAN1 mutations. Using different molecular genetic techniques, we identified 20 patients with a pathogenic or likely pathogenic SPTAN1 variant and reviewed their clinical, genetic and imaging data. SPTAN1 de novo alterations included seven unique missense variants and nine in-frame deletions/duplications of which 12 were novel. The recurrent three-amino acid duplication p.(Asp2303_Leu2305dup) occurred in five patients. Our patient cohort exhibited a broad spectrum of neurodevelopmental phenotypes, comprising six patients with mild to moderate intellectual disability, with or without epilepsy and behavioural disorders, and 14 patients with infantile epileptic encephalopathy, of which 13 had severe neurodevelopmental impairment and four died in early childhood. Imaging studies suggested that the severity of neurological impairment and epilepsy correlates with that of structural abnormalities as well as the mutation type and location. Out of seven patients harbouring mutations outside the α/β spectrin heterodimerization domain, four had normal brain imaging and three exhibited moderately progressive brain and/or cerebellar atrophy. Twelve of 13 patients with mutations located within the spectrin heterodimer contact site exhibited severe and progressive brain, brainstem and cerebellar atrophy, with hypomyelination in most. We used fibroblasts from five patients to study spectrin aggregate formation by Triton-X extraction and immunocytochemistry followed by fluorescence microscopy. αII/βII aggregates and αII spectrin in the insoluble protein fraction were observed in fibroblasts derived from patients with the mutations p.(Glu2207del), p.(Asp2303_Leu2305dup) and p.(Arg2308_Met2309dup), all falling in the nucleation site of the α/β spectrin heterodimer region. Molecular modelling of the seven SPTAN1 amino acid changes provided preliminary evidence for structural alterations of the A-, B- and/or C-helices within each of the mutated spectrin repeats. We conclude that SPTAN1-related disorders comprise a wide spectrum of neurodevelopmental phenotypes ranging from mild to severe and progressive. Spectrin aggregate formation in fibroblasts with mutations in the α/β heterodimerization domain seems to be associated with a severe neurodegenerative course and suggests that the amino acid stretch from Asp2303 to Met2309 in the α20 repeat is important for α/β spectrin heterodimer formation and/or αII spectrin function.

Abstract 2898: ST8SIA1 is up-regulated in triple negative breast cancer and its expression is positively correlated with TP53 mutations and a cancer stem cell gene signature

Abstract We discovered ganglioside GD2 as a breast cancer stem cell (BCSCs) marker in triple negative breast cancer (TNBC). GD2 biosynthesis is tightly regulated by the enzyme ST8SIA1 (GD3 synthase). However, expression of ST8SIA and its association with different breast cancer sub-types is not known. Here we hypothesize that ST8SIA1 is up-regulated in TNBC or “basal-type” breast tumors and associated with stemness in primary breast tumors. To investigate ST8SIA1 expression in primary tumors, we analyzed RNAseq data from the cancer genome atlas (TCGA) data base, which includes data from 1105 primary and metastatic breast tumors as well as adjacent normal tissues. We found that ST8SIA1 expression varied widely among different breast tumors. Interestingly, “basal-type” tumors expressed the highest levels of ST8SIA1 compared to all other types of breast cancers including luminal-A or luminal-B or HER2-enriched tumors (p < 0.01). In addition, TNBC (n = 115) expressed 4.6-fold higher ST8SIA1 levels compared to hormone receptors positive tumors including ER+ or PR+ or HER2+ tumors (n = 852, p<0.001). Survival analysis by log-rank test indicated that patients with ST8SIA1high tumors survive shorter (median survival 2.6 years) compared to patients with ST8SIA1low tumors (median survival 4.3 years). Next, we investigates the association between ST8SIA1expression and the most commonly mutated genes in breast cancer. We chose the top 20 most frequently mutated genes in TCGA dataset and examined their correlation with ST8SIA1 mRNA expression. We found that, among the top 20 mutations, TP53 had a very strong positive correlation with ST8SIA1 expression (p < 0.00001). In fact, the expression of ST8SIA1 was > 2-fold higher in TP53-mutated compared to wild type tumors. The other positively correlated mutation was a nuclear envelop protein called the spectrin repeat containing protein (nuclear envelope 1 or SYNE1; p < 0.05). Mutations in GATA3 were the most negatively correlated (p < 0.001) with ST8SIA1 expression. Interestingly, GATA3 plays a role in epithelial cell differentiation in the mammary gland, supporting the notion that ST8SIA1 is a stem cell-associated gene. In addition, correlation of ST8SIA1 mRNA expression with other genes revealed that FOXA1, the protein which is co-expressed with GATA3 and serves as negative predictor of “basal-type” of breast cancer, was down regulated in ST8SIA1high tumors. In conclusion, ST8SIA1 is associated with “basal-type” TNBC tumors and it has a strong positive correlation with TP53 mutations and negative correlation with GATA3 mutations. Knowing that TP53 mutations have a major role in tumorigenesis and drug resistance, these data suggest that ST8SIA1 could be a potential therapeutic target in patients with TP53-mutant TNBC. Citation Format: V. Lokesh Battula, Yuanqing Yan, Khoa Nguyen, Kim-Anh Do, Michael Andreeff. ST8SIA1 is up-regulated in triple negative breast cancer and its expression is positively correlated with TP53 mutations and a cancer stem cell gene signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2898. doi:10.1158/1538-7445.AM2017-2898

The Abl pathway bifurcates to balance Enabled and Rac signaling in axon patterning in Drosophila

The Abl tyrosine kinase signaling network controls cell migration, epithelial organization, axon patterning and other aspects of development. Although individual components are known, the relationships among them remain unresolved. We now use FRET measurements of pathway activity, analysis of protein localization and genetic epistasis to dissect the structure of this network in Drosophila We find that the adaptor protein Disabled stimulates Abl kinase activity. Abl suppresses the actin-regulatory factor Enabled, and we find that Abl also acts through the GEF Trio to stimulate the signaling activity of Rac GTPase: Abl gates the activity of the spectrin repeats of Trio, allowing them to relieve intramolecular repression of Trio GEF activity by the Trio N-terminal domain. Finally, we show that a key target of Abl signaling in axons is the WAVE complex that promotes the formation of branched actin networks. Thus, we show that Abl constitutes a bifurcating network, suppressing Ena activity in parallel with stimulation of WAVE. We suggest that the balancing of linear and branched actin networks by Abl is likely to be central to its regulation of axon patterning.

Abstract P6-07-26: ST8SIA1 is over-expressed in triple negative breast cancer and associated with p53 mutations

Abstract Cancer stem cells constitute a small fraction of cells present in primary tumors, are highly tumorigenic, drug resistant and cause metastases. We discovered Ganglioside GD2 as a breast cancer stem cell (BCSCs) marker in triple negative breast cancer (TNBC). GD2 biosynthesis is tightly regulated by the enzyme ST8SIA1 (GD3 synthase). However, expression of ST8SIA and its association with different breast cancer sub-types is not known. Here we hypothesize that ST8SIA1 is up-regulated in TNBC or basal-type breast tumors and associated with stemness in primary breast tumors. Frist, we examined ST8SIA1 protein expression in breast cancer cell lines (n=12) and found up-regulation of ST8SIA1 in basal compared to luminal-type breast cancer cells. To investigate ST8SIA1's expression in primary tumors, we analyzed RNAseq data from the cancer genome atlas (TCGA) data base which has expression data from 1105 primary and metastatic breast tumors as well as adjacent normal tissues. We found that ST8SIA1 expression varies widely among different breast tumors and that 'basal' type tumors express highest levels of ST8SIA1 compared to all other types of breast cancers including luminal-A or luminal-B or HER2 enriched (p<0.01) tumors. In addition, triple negative breast cancer (TNBC, n=115) express 4.63fold higher ST8SIA1 levels compared to hormone receptors positive tumors including ER+ or PR+ or HER2+ tumors (n=852, p<0.001). Survival analysis by log-rank test indicates that patients with ST8SIA1high tumors survive shorter (median survival 2.6 years) compared to patients with ST8SIA1low tumors (median survival 4.3 years). These data suggests that ST8SIA1 expression is associated with basal-like TNBC tumor types and high ST8SIA1 levels in tumors may shorten survival in TNBC patients. To investigate the association between ST8SIA1expression and the most commonly mutated genes in breast cancer, we chose the top 20 most frequently mutated genes in TCGA dataset and examined their correlation with ST8SIA1 mRNA expression. We found that, among the top 20 mutations, p53 has a very strong positive correlation with ST8SIA1 expression (p<0.00001). The expression of ST8SIA1 is >2 fold higher in p53 mutated tumors compared to p53 wild type tumors. The other positively correlated mutation was a nuclear envelop protein called Spectrin Repeat Containing, Nuclear Envelope 1 (SYNE1; p<0.05). Mutations in GATA3 are the most negatively correlated (p<0.001) with ST8SIA1 expression. Interestingly, GATA3 plays a role in epithelial cell differentiation in the mammary gland, supporting the notion that ST8SIA1 is a stem cell-associated gene. In addition, correlation of ST8SIA1 mRNA expression with other genes revealed that FOXA1, the protein which is co-expressed with GATA3 and serves as negative predictor of basal type of breast cancer, is down regulated in ST8SIA1high tumors. In conclusion, ST8SIA1 is associated with basal-type TNBC tumors and has a strong positive correlation with p53 mutations and negative correlation with GATA3 mutations. Knowing that TP53 mutations have a major role in tumorigenesis and drug resistance, these data suggest ST8SIA1 as a potential therapeutic target in TNBC patients with p53 mutations. Citation Format: Yan Y, Nguyen K, Do K-A, Ueno N, Andreeff M, Battula VL. ST8SIA1 is over-expressed in triple negative breast cancer and associated with p53 mutations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-26.

Simulating Force Response at Cellular Junctions: Desmoplakin as a Molecular Force Sensor?

Desmosomes are cell-cell junctions with a key role in mechano-sensation. However, it is currently unknown how the desmosomal complex and its proteins respond to mechanical force, transmitting chemical signals. We study desmoplakin, one of the obligate desmosomal proteins, identifying its possible role in mechanosensing. The most striking feature of this protein is the presence of an SH3 domain embedded in one of its six spectrin repeat domains. Intriguingly, the spectrin repeats hide the typical SH3 binding site, suggesting that either (i) the SH3 domain serves as a stabilizer of the protein through its interaction with one of the spectrin repeats, or (ii) it is activated only under force, initiating a signaling cascade for mechanosensing. To explore these alternatives, we performed equilibrium as well as force-probe Molecular Dynamics simulations on the central region of desmoplakin. To bridge towards physiologically relevant time scales, we repeated our simulations through a wide range of pulling velocities with a total simulation time of several tens of microseconds. We find desmoplakin to feature a clear sawtooth-like force profile, with each peak corresponding to modular spectrin repeat unfolding events followed by the final SH3 unfolding. The SH3 domain not only stabilizes desmoplakin, but also remains intact until the very last part of the trajectory, allowing interactions with downstream partners. Interestingly, we find the same behavior in the homologous plectin protein, suggesting this dual role of the SH3 domain to be conserved throughout the plakin family of proteins. However, plectin unfolds at significantly lower forces, involving an early or forceless loss of the SH3 domain-spectrin repeat interface. We identify specific mutations that promote this unfolding pathway, explaining the evolutionary adaptation of the cytosolic compared to the desmosomal mechano-sensing molecule. Our results suggest a direct role of unique SH3-insertion in spectrin repeat proteins in cellular mechanotransduction, and will be validated by direct comparison to single-molecule force spectroscopy experiments carried out by our collaborators.

Mechanical Response of Nesprin-2G to Cytoskeletal Forces Regulates Linc Complex-Dependent Mechanotransduction

Nuclear geometry and positioning are affected by cytoskeletal forces, which plays an important role in cellular mechanotrasnduction. The LINC (LInkers of the Nucleoskeleton and Cytoskeleton) complex has been shown to bear mechanical load and mediate force transmission from the cytoskeleton into the nucleus, however, molecular mechanisms of this phenomenon are not yet understood. Giant nesprin-2 (nesprin-2G) forms a direct linkage between the actin cytoskeleton and SUN proteins inside the perinuclear space. These linkers are shown to be under constant tension especially in the apical and equatorial planes of the nucleus and have 56 spectrin repeats along their rod domain. A functional construct of nespring-2G containing only four spectrin repeats, referred to as mini-nesprin-2G, was shown to rescue the nuclear movement defects observed in nesprin-2G-depleted fibroblasts. Since it is intrinsically difficult to measure forces and track residue-level interactions across individual proteins using current experimental techniques, we used molecular dynamics simulations to study mechanical response of nesprin molecules to cytoskeletal forces. Mini-nesprins are excellent candidates for this purpose as their small size allows for long simulation periods necessary for capturing important molecular events. Our results provides a detailed understanding of how mechanical modes of mini-nesprin-2G are excited and contribute to its response to various cytoskeletal forces around the nucleus. Also, we simulated force-induced conformational changes of the Actin Binding Domain (ABD) of mini-nesprin-2G, which are most likely induced by the axial tension along the actin bundles at the apical plane of the nucleus. Furthermore, we examined the effect of ABD conformation on its actin binding affinity by comparing the strength of interactions between distinct conformations of the ABD domain of mini-nesprin-2G and actin. Lastly, we used force distribution analysis to identify force-sensitive residues along the mini-nesprin-2G structure and tested the effect of mutating them to alanine on transferring forces to the nucleus.

Identification of Genomic Regions Related to pH in Nellore Beef Cattle

ObjectivesThe aim of this study was to identify genomic regions that potentially have association with pH in Nellore cattle.Materials and MethodsPost mortem muscle metabolism (pH) was measured on samples of Longissimus thoracis muscle collected 24 h post mortem using a portable pHmeter (Hanna HI9963) from 1208 feedlot Nellore steers slaughthered at 2 yr of age. Genotypic data of all animals was used for genome wide association studies through the ssGWAS method. Those animals were genotyped with the Illumina Bovine beadchip HD GGPi (74K). Based on another Nellore population genotyped for Illumina beadchip BovineHD (777K), genotypes were imputed using the FImpute software. Analyses were performed using a pedigree composed by 6276 animals. Single step analyses were performed using the Blupf90 family of programs considering windows of 10 markers to estimate the SNP effects. This procedure enables the identification of regions associated with pH along the chromosomes. After quality control (MAF < 0.05%, call rate < 90%), 463,995 SNPs in autosomal chromosomes were used in the association analyses.ResultsThe current investigation revelead 15 regions located in 10 different chromosomes (2, 3, 4, 8, 9, 11, 12, 16, 17, and 21), which explained more than 1% of the additive variance (Fig. 1). Gene identification was performed using the BioMart do Ensembl Genome Browser tool (www.ensembl.org). The following genes were identified in 6 distinct regions: WD repeat, sterile ɑ motif and U-box domain containing 1; palmdelphin; insulin like growth factor binding protein 1; Bos taurus charged multivesicular body protein 7; tumor necrosis factor receptor superfamily, member 10d, decoy with truncated death domain; rho-related BTB domain-containing protein 2; spectrin repeat containing nuclear envelope protein 1 e ubiquitin specific peptidase like 1.ConclusionThe ssGWAS method, using high density panel, allowed the identification of regions and genes related to pH in Nellore beef cattle. Further investigation combining information of the reported genes and its biological pathways is required to better evaluate their importance for meat quality traits.

The Abl pathway bifurcates to balance Enabled and Rac signaling in axon patterning in Drosophila.

The Abl tyrosine kinase signaling network controls cell migration, epithelial organization, axon patterning and other aspects of development. Although individual components are known, the relationships among them remain unresolved. We now use FRET measurements of pathway activity, analysis of protein localization and genetic epistasis to dissect the structure of this network in Drosophila We find that the adaptor protein Disabled stimulates Abl kinase activity. Abl suppresses the actin-regulatory factor Enabled, and we find that Abl also acts through the GEF Trio to stimulate the signaling activity of Rac GTPase: Abl gates the activity of the spectrin repeats of Trio, allowing them to relieve intramolecular repression of Trio GEF activity by the Trio N-terminal domain. Finally, we show that a key target of Abl signaling in axons is the WAVE complex that promotes the formation of branched actin networks. Thus, we show that Abl constitutes a bifurcating network, suppressing Ena activity in parallel with stimulation of WAVE. We suggest that the balancing of linear and branched actin networks by Abl is likely to be central to its regulation of axon patterning.

Trio, a Rho Family GEF, Interacts with the Presynaptic Active Zone Proteins Piccolo and Bassoon.

Synaptic vesicles (SVs) fuse with the plasma membrane at a precise location called the presynaptic active zone (AZ). This fusion is coordinated by proteins embedded within a cytoskeletal matrix assembled at the AZ (CAZ). In the present study, we have identified a novel binding partner for the CAZ proteins Piccolo and Bassoon. This interacting protein, Trio, is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) known to regulate the dynamic assembly of actin and growth factor dependent axon guidance and synaptic growth. Trio was found to interact with the C-terminal PBH 9/10 domains of Piccolo and Bassoon via its own N-terminal Spectrin repeats, a domain that is also critical for its localization to the CAZ. Moreover, our data suggest that regions within the C-terminus of Trio negatively regulate its interactions with Piccolo/Bassoon. These findings provide a mechanism for the presynaptic targeting of Trio and support a model in which Piccolo and Bassoon play a role in regulating neurotransmission through interactions with proteins, including Trio, that modulate the dynamic assembly of F-actin during cycles of synaptic vesicle exo- and endocytosis.

Identification of novel nesprin-1 binding partners and cytoplasmic matrin-3 in processing bodies.

Nesprins are highly conserved spectrin repeat-containing scaffold proteins predominantly known to function at the nuclear envelope (NE). However, nesprin isoforms are emerging with localizations and scaffolding functions at sites away from the NE, suggesting their functions are more diverse than originally thought. In this study, we combined nesprin-1 coimmunoprecipitations with mass spectrometry to identify novel nesprin-1 binding partners for isoforms that localize to subcellular compartments beyond the NE. We show that one of these interactors, matrin-3 (matr3), localizes to mRNA processing bodies (PBs), where we have previously shown a nesprin-1 isoform to localize. Furthermore, we show that Matr3 is part of PB mRNP complexes, is a regulator of miRNA-mediated gene silencing, and possibly shuttles to stress granules in stressed cells. More importantly, we identify a new C-terminally truncated Matr3 isoform that is likely to be involved in these functions and PB localization. This study highlights several novel nesprin-1 binding partners and a new function and localization for Matr3 in cytoplasmic RNA granules.

Cytoskeletal Integrators: The Spectrin Superfamily.

This review discusses the spectrin superfamily of proteins that function to connect cytoskeletal elements to each other, the cell membrane, and the nucleus. The signature domain is the spectrin repeat, a 106-122-amino-acid segment comprising three α-helices. α-actinin is considered to be the ancestral protein and functions to cross-link actin filaments. It then evolved to generate spectrin and dystrophin that function to link the actin cytoskeleton to the cell membrane, as well as the spectraplakins and plakins that link cytoskeletal elements to each other and to junctional complexes. A final class comprises the nesprins, which are able to bind to the nuclear membrane. This review discusses the domain organization of the various spectrin family members, their roles in protein-protein interactions, and their roles in disease, as determined from mutations, and it also describes the functional roles of the family members as determined from null phenotypes.

Characterization of novel dystonia musculorum mutant mice: Implications for central nervous system abnormality

We identified a novel spontaneous mutant mouse showing motor symptoms that are similar to those of the dystonia musculorum (dt) mouse. The observations suggested that the mutant mice inherited the mild dt phenotype as an autosomal recessive trait. Linkage analysis showed that the causative gene was located near D1Mit373 and D1Mit410 microsatellite markers on chromosome 1, which are close to the dystonin (Dst) gene locus. To investigate whether Dst is the causative gene of the novel mutant phenotype, we crossed the mutant with Dst gene trap (DstGt) mice. Compound heterozygotes showed a typical dt phenotype with sensory degeneration and progressive motor symptoms. DNA sequencing analysis identified a nonsense mutation within the spectrin repeats of the plakin domain. The novel mutant allele was named dt23Rbrc. Motor abnormalities in homozygous dt23Rbrc/dt23Rbrc mice are not as severe as homozygous DstGt/DstGt mice. Histological analyses showed abnormal neurofilament (NF) accumulation in the nervous system of homozygous dt23Rbrc/dt23Rbrc mice, which is characteristic of the dt phenotype. We mapped the distribution of abnormal NF-accumulated neurons in the brain and found that they were located specifically in the brainstem, spinal cord, and in regions such as the vestibular nucleus, reticular nucleus, and red nucleus, which are implicated in posture and motor coordination pathways. The quantification of abnormal NF accumulation in the cytoplasm and spheroids (axons) of neurons showed that abnormal NF immunoreactivity was lower in homozygous dt23Rbrc/dt23Rbrc mice than in homozygous DstGt/DstGt mice. Therefore, we have identified a novel hypomorphic allele of dt, which causes histological abnormalities in the central nervous system that may account for the abnormal motor phenotype. This novel spontaneously occurring mutant may become a good model of hereditary sensory and autonomic neuropathy type 6, which is caused by mutations in the human DST gene.

The Structure of the Plakin Domain of Plectin Reveals an Extended Rod-like Shape

Plakins are large multi-domain proteins that interconnect cytoskeletal structures. Plectin is a prototypical plakin that tethers intermediate filaments to membrane-associated complexes. Most plakins contain a plakin domain formed by up to nine spectrin repeats (SR1-SR9) and an SH3 domain. The plakin domains of plectin and other plakins harbor binding sites for junctional proteins. We have combined x-ray crystallography with small angle x-ray scattering (SAXS) to elucidate the structure of the plakin domain of plectin, extending our previous analysis of the SR1 to SR5 region. Two crystal structures of the SR5-SR6 region allowed us to characterize its uniquely wide inter-repeat conformational variability. We also report the crystal structures of the SR7-SR8 region, refined to 1.8 Å, and the SR7-SR9 at lower resolution. The SR7-SR9 region, which is conserved in all other plakin domains, forms a rigid segment stabilized by uniquely extensive inter-repeat contacts mediated by unusually long helices in SR8 and SR9. Using SAXS we show that in solution the SR3-SR6 and SR7-SR9 regions are rod-like segments and that SR3-SR9 of plectin has an extended shape with a small central kink. Other plakins, such as bullous pemphigoid antigen 1 and microtubule and actin cross-linking factor 1, are likely to have similar extended plakin domains. In contrast, desmoplakin has a two-segment structure with a central flexible hinge. The continuous versus segmented structures of the plakin domains of plectin and desmoplakin give insight into how different plakins might respond to tension and transmit mechanical signals.

EGFR and SYNE2 are associated with p21 expression and SYNE2 variants predict post-operative clinical outcomes in HBV-related hepatocellular carcinoma.

This study was to explore the association between gene variants and p21 expression and investigate the TP53-independent p21 regulation in hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) patients from Guangxi by genome-wide association study. 426 HBV-related HCC patients were enrolled. Results showed that, after quality control, a total of 21,643 SNPs were identified in 107 p21 positive and 298 p21 negative patients. The variants of epidermal growth factor receptor (EGFR; rs2227983 and rs6950826) and spectrin repeat containing, nuclear envelope 2 (SYNE2; rs8010699, rs4027405 and rs1890908) were associated with p21 expression. Moreover the haplotype block (rs2227983 and rs6950826, r2 = 0.378) in EGFR and the haplotype block in SYNE2 (rs8010699 was in strong LD with rs4027405 and rs1890908 (r2 = 0.91 and 0.70, respectively)) were identified, and the haplotype A-G of EGFR and haplotype G-A-A of SYNE2 were significantly associated with p21 expression (P < 0.01). rs4027405 and rs1890908 were significantly associated with overall survival, and patients with AG/GG genotypes of SYNE2 gene had a worse overall survival (P = 0.001, P = 0.002). Our findings indicate that variants of EGFR and SYNE2 play an important role in p21 regulation and are associated with the clinical outcome of HBV-related HCC in a TP53-indenpdent manner.

Characterisation of Schizosaccharomyces pombe α-actinin.

The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, α-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of α-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional α-actinin, we have cloned and characterized each structural domain. Our results show that this a-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional α-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other α-actinins, which may reduce the affinity for actin.

Hydrophobic Interactions Elicit Cooperative Response in Dystrophin

The primary role of dystrophin, a 427 kDa elongated protein, is to stabilize the membrane of muscle cells against the mechanical forces deriving from muscle contraction and relaxation; its absence or mutation lead to various forms of muscular dystrophy. Dystrophin binds to actin through its N-terminus actin binding domain (ABD1), and connects to the cell membrane through its C-terminus; in between, 24 spectrin repeats (SR) with interspersed hinges provide structural flexibility and have been proposed to actively dissipate the mechanical stress. Yet, dystrophin's mechanism of function is largely unknown, preventing effective drug development. Our working hypothesis is that dystrophin's domains are allosterically coupled through specific hydrophobic interactions, and that the response to mechanical stress is propagated across domains through order-to-disorder transitions at their interface. We test this hypothesis with all-atom molecular dynamics simulations of dystrophin's SR 1 monomer, SR 17-18 dimer, and the ABD1 domain. Our results support the hypothesis by showing that (a) forced unfolding by pulling initiates at the interface between SR repeats; (b) the linker between SR 17-18 is flexible and allows for distinct conformations at the interface between domains, characterized by different hydrophobic interaction patterns; (c) mutual information analyses show that conformational changes at the dimer's interface and termini are coupled; and (d) hydrophobic interactions between the two CH domains and the connecting linker stabilize a closed conformation of ABD1 that is alternative to the open crystal structure and explains experimental fluorescence results. Our results suggest that the disease may originate from altered hydrophobic interactions, and pave the way for targeted drug development.

Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting.

Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins.