Splice variants of Enigma homolog, differentially expressed during heart development, promote or prevent hypertrophy

Aims Proteins with a PDZ (for PSD-95, DLG, ZO-1) and one to three LIM (for Lin11, Isl-1, Mec-3) domains are scaffolding sarcomeric and cytoskeletal elements that form structured muscle fibres and provide for the link to intracellular signalling by selectively associating protein kinases, ion channels, and transcription factors with the mechanical stress-strain sensors. Enigma homolog (ENH) is a PDZ-LIM protein with four splice variants: ENH1 with an N-terminal PDZ domain and three C-terminal LIM domains and ENH2, ENH3, and ENH4 without LIM domains. We addressed the functional role of ENH alternative splicing. Methods and results We studied the expression of the four ENH isoforms in the heart during development and in a mouse model of heart hypertrophy. All four isoforms are expressed in the heart but the pattern of expression is clearly different between embryonic, neonatal, and adult stages. ENH1 appears as the embryonic isoform, whereas ENH2, ENH3, and ENH4 are predominant in adult heart. Moreover, alternative splicing of ENH was changed following induction of heart hypertrophy, producing an ENH isoform pattern similar to that of neonatal heart. Next, we tested a possible causal role of ENH1 and ENH4 in the development of cardiac hypertrophy. When overexpressed in rat neonatal cardiomyocytes, ENH1 promoted the expression of hypertrophy markers and increased cell volume, whereas, on the contrary, ENH4 overexpression prevented these changes. Conclusion Antagonistic splice variants of ENH may play a central role in the adaptive changes of the link between mechanical stress-sensing and signalling occurring during embryonic development and/or heart hypertroph

Structural basis of Sec-independent membrane protein insertion by YidC

[プレスリリース]バイオサイエンス研究科膜分子複合機能学研究室の塚崎智也准教授らの研究グループが、タンパク質を細胞膜に組み込むメカニズムを解明しました（2014/04/17）Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively1, 2. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG3, 4, 5. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin

X線自由電子レーザーを用いて、光照射によるチャネルロドプシンの構造変化の過程を捉えることに成功. 京都大学プレスリリース. 2021-03-26.Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore

Functions of fasciculation and elongation protein zeta-1

Fasciculation and elongation protein zeta-1 (FEZ1) is a mammalian ortholog of the Caenorhabditis elegans UNC-76 protein that possesses four coiled-coil domains and a nuclear localization signal. It is mainly expressed in the brain. Suppression of FEZ1 expression in cultured embryonic neurons causes deficiency of neuronal differentiation. Recently, proteomic techniques revealed that FEZ1 interacts with various intracellular partners, such as signaling, motor, and structural proteins. FEZ1 was shown to act as an antiviral factor. The findings reported so far indicate that FEZ1 is associated with neuronal development, neuropathologies, and viral infection. Based on these accumulating evidences, we herein review the biological functions of FEZ1. KEYWORDS: FEZ1, neuronal differentiation, neuronal disorders, virus infection, organelle transport DISCOVERY OF FASCICULATION AND ELONGATION PROTEIN ZETA-1 (FEZ1) FEZ1 is a mammalian ortholog of UNC-76, a protein found in the nematode Caenorhabditis elegans. UNC-76 has been used in an attempt to elucidate the mechanisms of locomotory defects. Genetic screenings of C. elegans mutants showing locomotory defects (uncoordinated or unc mutants) allowed the identification of various genes related to deficiencies in axonal guidance. Based on such screening, at least two groups of genes were found to be necessary for axonal elongation in fascicles The C. elegans unc-76 mutant exhibits two types of axonal defects in fascicles MOLECULAR ORGANIZATION AND EXPRESSION PROFILE OF FEZ1 In the human genome, the FEZ1 gene is located on chromosome 11. The human FEZ1 gene encodes a protein of 392 amino acids with a molecular mass of 45 kDa, showing ~35% identity with UNC-76. Maturana et al.: Functions of FEZ1 Protein TheScientificWorldJOURNAL (2010) 10, 1646-1654 1648 FIGURE 2. Functions of FEZ1 based on its binding partners. Details are described in the text. In addition to multiple coiled-coil domains, the carboxyl (C)-terminal half-region of FEZ1 contains a nuclear localization signal (NLS) In adult rats, FEZ1 mRNA is exclusively expressed in the brain. In particular, abundant expression is seen in the mitral and granular cells of the olfactory bulb, the granule cells of the dentate gyrus region CA1-3 in the hippocampus (highest expression) When expressed in human embryonic kidney HEK293 cells, FEZ1 is localized not only in the cytoplasm, but also in the nucleus BINDING PARTNERS AND BIOLOGICAL FUNCTIONS OF FEZ1 The biological functions of FEZ1 would be involved in the development and function of the central nervous system because of its brain-specific expression. Using proteomic approaches (particularly yeast two-hybrid screening assays), various functional proteins have been identified to interact with FEZ1, including factors that have roles in the cytoskeleton network, transport of cargoes, regulation of the cell cycle, and retrovirus propagation. Thus, the diversity of these binding partners implies that FEZ1 is a multirole molecule in neuronal development, neuropathologies, and neuronal resistance to viral infection Neuronal Differentiation through Phosphorylation by PKC FEZ1 was identified as a new PKC-interacting protein Regulation of Anterograde Axonal Transport by the Interaction with the Kinesin Superfamily In an attempt to discover new interacting proteins for Drosophila motor protein kinesin-1, the C-terminus of kinesin heavy chain (KHC) was identified to interact with Drosophila UNC-76 by genetic analysis Maturana et al.: Functions of FEZ1 Protein TheScientificWorldJOURNAL (2010) 10, 1646-1654 1650 Stabilizing and Extension of the Microtubule Network FEZ1 associates with α-and β-tubulin, which comprise the microtubules in extending neuronal axons Centrosomal Organization by Association with Centrosomal Proteins Recently, FEZ1 was found to interact with never in mitosis gene A (NIMA)-related kinase 1 (NEK1) FEZ1 in Neuronal Disorders FEZ1 was found to be an interacting partner of the schizophrenia-related protein disrupted in schizophrenia 1 (DISC1) More recently, FEZ1-deficient mice were generated Antiviral Infection Activity Interestingly, FEZ1 was identified as an interacting partner of agnoprotein, a small protein composed of 71 amino acids and encoded in the JC virus (JCV) genome Intrinsic immunity (an antiviral system of host cells) is mediated by various intracellular factors that specifically block the infection of eukaryotic retroviruses, such as tripartite motif 5α (TRIM5α and apolipoprotein B mRNA-editing catalytic polypeptide (APOBEC). Both proteins are responsible for the intrinsic immunity of various cell types (particularly neurons) The brain is one of the main targets for human immunodeficiency virus type 1 (HIV-1). Most patients infected with HIV-1 are subject to neurological disorders, such as cognitive impairment and HIVassociated dementia Other Functions Yeast two-hybrid screening of a human fetal brain cDNA library identified 16 FEZ1-interacting proteins FEZ2 A homolog of FEZ1 (named FEZ2) was first found by Bloom and Horvitz 1652 PKC induces the spontaneous neuronal differentiation of PC12 cells CONCLUSIONS AND FUTURE PERSPECTIVES Despite its restricted expression in the brain, FEZ1 can associate with various proteins at different locations in neurons. The possible functions of FEZ1 are summarized i

Functions of Fasciculation and Elongation Protein Zeta-1 (FEZ1) in the Brain

Fasciculation and elongation protein zeta-1 (FEZ1) is a mammalian ortholog of the Caenorhabditis elegans UNC-76 protein that possesses four coiled-coil domains and a nuclear localization signal. It is mainly expressed in the brain. Suppression of FEZ1 expression in cultured embryonic neurons causes deficiency of neuronal differentiation. Recently, proteomic techniques revealed that FEZ1 interacts with various intracellular partners, such as signaling, motor, and structural proteins. FEZ1 was shown to act as an antiviral factor. The findings reported so far indicate that FEZ1 is associated with neuronal development, neuropathologies, and viral infection. Based on these accumulating evidences, we herein review the biological functions of FEZ1

Molecular dynamics of channelrhodopsin at the early stages of channel opening

Channelrhodopsin (ChR) is a light-gated cation channel that responds to blue light. Since ChR can be readily expressed in specific neurons to precisely control their activities by light, it has become a powerful tool in neuroscience. Although the recently solved crystal structure of a chimeric ChR, C1C2, provided the structural basis for ChR, our understanding of the molecular mechanism of ChR still remains limited. Here we performed electrophysiological analyses and all-atom molecular dynamics (MD) simulations, to investigate the importance of the intracellular and central constrictions of the ion conducting pore observed in the crystal structure of C1C2. Our electrophysiological analysis revealed that two glutamate residues, Glu122 and Glu129, in the intracellular and central constrictions, respectively, should be deprotonated in the photocycle. The simulation results suggested that the deprotonation of Glu129 in the central constriction leads to ion leakage in the ground state, and implied that the protonation of Glu129 is important for preventing ion leakage in the ground state. Moreover, we modeled the 13-cis retinal bound; i.e., activated C1C2, and performed MD simulations to investigate the conformational changes in the early stage of the photocycle. Our simulations suggested that retinal photoisomerization induces the conformational change toward channel opening, including the movements of TM6, TM7 and TM2. These insights into the dynamics of the ground states and the early photocycle stages enhance our understanding of the channel function of ChR

Differential gene expression in well-regulated and dysregulated pancreatic beta-cell (MIN6) sublines

To identify genes involved in regulated insulin secretion, we have established and characterized two sublines derived from the mouse pancreatic beta-cell line MIN6, designated B1 and C3. They have a similar insulin content, but differ in their secretory properties. B1 responded to glucose in a concentration- and cell confluence-dependent manner, whereas C3 did not. B1 cells were stimulated by phorbol 12-myristate 13-acetate, leucine, arginine, glibenclamide, isobutylmethylxanthine, and KCl, whereas C3 did not respond (leucine, arginine, and glibenclamide) or responded to a lesser extent (isobutylmethylxanthine, phorbol 12-myristate 13-acetate, and KCl). Although intracellular Ca(2+) rose in response to glucose in B1 but not C3 cells, KCl increased intracellular Ca(2+) in a similar manner in both sublines. GLUT-1, GLUT-2, Kir6.2, and SUR1 expression was not significantly different between B1 and C3 cells, whereas E-cadherin was more abundantly expressed in B1 cells. A more complete list of differentially expressed genes was established by suppression subtractive hybridization and high density (Affymetrix) oligonucleotide microarrays. Genes were clustered according to known or putative function. Those involved in metabolism, intracellular signaling, cytoarchitecture, and cell adhesion are of potential interest. These two sublines should be useful for identification of the genes and mechanisms involved in regulated insulin secretion of the pancreatic beta-cell