Migration defects by DISC1 knockdown in C57BL/6, 129X1/SvJ, and ICR strains via in utero gene transfer and virus-mediated RNAi

Disrupted-in-Schizophrenia 1 (DISC1) is a promising genetic risk factor for major mental disorders. Many groups repeatedly reported a role for DISC1 in brain development in various strains of mice and rats by using RNA interference (RNAi) approach. Nonetheless, due to the complexity of its molecular disposition, such as many splice variants and a spontaneous deletion in a coding exon of the DISC1 gene in some mouse strains, there have been debates on the interpretation on these published data. Thus, in this study, we address this question by DISC1 knockdown via short-hairpin RNAs (shRNAs) against several distinct target sequences with more than one delivery methodologies into several mouse strains, including C57BL/6, ICR, and 129X1/SvJ. Here, we show that DISC1 knockdown by in utero electroporation of shRNA against exons 2, 6, and 10 consistently results in neuronal migration defects in the developing cerebral cortex, which are successfully rescued by co-expression of full-length DISC1. Furthermore, lentivirus-mediated shRNA also led to migration defects, which is consistent with two other methodologies already published, such as plasmid-mediated and retrovirus-mediated ones. The previous study by Song’s group also reported that, in the adult hippocampus, the phenotype elicited by DISC1 knockdown with shRNA targeting exon 2 was consistently seen in both C57BL/6 and 129S6 mice. Taken together, we propose that some of DISC1 isoforms that are feasible to be knocked down by shRNAs to exon 2, 6, and 10 of the DISC1 gene play a key role for neuronal migration commonly in various mouse strains and rats.     

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple‐mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two‐orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross‐reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr‐containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.     

The aspartic acid of Fyn at 390 is critical for neuronal migration during corticogenesis

The mammalian cerebral cortex develops through the coordinated migration of postmitotic neurons. Fyn, a member of the Src tyrosine kinase family (SFKs), is involved in the neuronal migration and the absence of Fyn leads to abnormal migration. However, the molecular mechanism whereby Fyn acts on migrating neurons has remained unclear. Here, we employed two Fyn mutants (Fyn259T and FynD390A) to investigate the function of Fyn kinase domain in neuronal migration. Using in utero electroporation, we co-transfected the migrating neurons in embryonic cortex with these mutants combined with plasmid expressing GFP. Interestingly, although both of them impaired neuronal migration, FynD390A, rather than Fyn259T, induced remarkable morphology change. Our work provides in vivo and in vitro evidence that the aspartic acid of Fyn at 390 is indispensable for the radial migration, and it is required for precise cooperation with focal adhesion kinase.     

Solution structure of N-terminal SH3 domain of Vav and the recognition site for Grb2 C-terminal SH3 domain

The three-dimensional structure of the N-terminal SH3 domain (residues 583–660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies.     

Diversity in peptide recognition by the SH2 domain of SH2B1

SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein‐protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate‐1 and ?2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C‐terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high‐resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand‐binding interface of SH2B1 with high specificity.     

Pax6 regulates regional development and neuronal migration in the cerebral cortex

Mutations in the Pax6 gene disrupt telencephalic development, resulting in a thin cortical plate, expansion of proliferative layers, and the absence of the olfactory bulb. The primary defect in the neuronal cell population of the developing cerebral cortex was analysed by using mouse chimeras containing a mixture of wild-type and Pax6-deficient cells. The chimeric analysis shows that Pax6 influences cellular activity throughout corticogenesis. At early stages, Pax6-deficient and wildtype cells segregate into exclusive patches, indicating an inability of different cell genotypes to interact. At later stages, cells are sorted further based on telencephalic domains. Pax6-deficient cells are specifically reduced in the mediocaudal domain of the dorsal telencephalon, indicating a role in regionalization. In addition, Pax6 regulates the process of radial migration of neuronal precursors. Loss of Pax6 particularly affects movement of neuronal precursors at the subventricular zone/intermediate zone boundary at a transitional migratory phase essential for entry into the intermediate zone. We suggest that the primary role of Pax6 is the continual regulation of cell surface properties responsible for both cellular identity and radial migration, defects of which cause regional cell sorting and abnormalities of migration in chimeras.     

The purinergic P2Y14 receptor axis is a molecular determinant for organism survival under in utero radiation toxicity

In utero exposure of the embryo and fetus to radiation has been implicated in malformations or fetal death, and often produces lifelong health consequences such as cancers and mental retardation. Here we demonstrate that deletion of a G-protein-coupled purinergic receptor, P2Y₁₄, confers potent resistance to in utero radiation. Intriguingly, a putative P2Y₁₄ receptor ligand, UDP-glucose, phenocopies the effect of P2Y₁₄ deficiency. These data indicate that P2Y₁₄ is a receptor governing in utero tolerance to genotoxic stress that may be pharmacologically targeted to mitigate radiation toxicity in pregnancy.     

Implication of SH2B1 gene polymorphism studies in gestational diabetes mellitus in Saudi pregnant women

Genome-wide association studies have identified loci that are firmly associated with obesity. The Src-homology-2 B adaptor protein 1 (SH2B1) loci is abundantly expressed in the brain, liver, heart, muscle, and fat tissues. Gestational diabetes mellitus (GDM) is a growing health concern that usually appears during the latter half of pregnancy, and it is characterized by carbohydrate intolerance of variable severity. The SH2B1 gene polymorphism has been linked with an increased risk of weight gain in several but not all population studies. This study aimed to investigate the genetic association of rs4788102 variants in the SH2B1 gene with GDM in Saudi pregnant women. Genomic DNA samples from 200 women with GDM and 300 women without GDM were genotyped using the TaqMan method. The distribution of the GG, GA, and AA genotypes was significantly different between GDM and non-GDM women (p < 0.05). Thus, we identified rs4788102 variants as additional risk factors for GDM in Saudi women, and we suggest that these variants may have a prognostic value.     

The SH3 domain of Src can downregulate its kinase activity in the absence of the SH2 domain-pY527 interaction

The contact between the SH2 domain and the C-terminal tail of c-Src inhibits its kinase activity via a complex network of interactions, including the SH3 domain. We examined the role of the SH3 domain in v-Src, where the C-terminal tail is mutated and unbound. We used the v-Src variants Prague C (PRC) and Schmidt-Ruppin A (SRA), which are of low and high kinase activities, respectively, to measure phosphorylation in vitro by immunoprecipitated kinases produced in Saccharomyces cerevisiae. Swapping the regulatory domains between SRA and PRC revealed that N117D, I96T, and V124L mutations in the n-src- and RT-loops of the SH3 domain of PRC are responsible for the low kinase activity of PRC. Moreover, introducing D117N, R95W, T96I, and L124V into activated c-Src(Y527F) caused a 2.5-fold increase in its activity. The mutations in the CD linker KP249,250DG and L255A, which were shown to activate c-Src, had no effect on the activity of the "SH2-activated" Src kinases. Together our data suggest that in the "SH2-activated" forms of Src, the SH3 domain continues to influence the kinase activity via the direct contacts of the n-src- and RT-loops with the kinase N-terminal lobe.     

The helically extended SH3 domain of the T cell adaptor protein ADAP is a novel lipid interaction domain

Adhesion and degranulation-promoting adapter protein (ADAP) is critically involved in downstream signalling events triggered by the activation of the T cell receptor. Cytokine production, proliferation and integrin clustering of T cells are dependent on ADAP function, but the molecular basis for these processes is poorly understood. We now show the hSH3 domain of ADAP to be a lipid-interaction module that binds to acidic lipids, including phosphatidylinositides. Positively charged surface patches of the domain preferentially bind to polyvalent acidic lipids such as PIP2 or PIP3 over the monovalent PS phospholipid and this interaction is dependent on the N-terminal helix of the hSH3 domain fold. Basic amino acid side-chains from the SH3 scaffold also contribute to lipid binding. In the context of T cell signalling, our findings suggest that ADAP, upon recruitment to the cell-cell junction as part of a multiprotein complex, directly interacts with phosphoinositide-enriched regions of the plasma membrane. Furthermore, the ADAP lipid interaction defines the helically extended SH3 scaffold as a novel member of membrane interaction domains.     

Molecular details of Itk activation by prolyl isomerization and phospholigand binding: the NMR structure of the Itk SH2 domain bound to a phosphopeptide

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a critical component of the regulatory apparatus controlling the activity of this immunologically important enzyme. To gain insight into the structural features associated with the activated form of Itk, we have solved the NMR structure of the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed changes in trans-hydrogen bond scalar couplings ((3h)J(NC')) that result from pY binding. Isomerization of a single prolyl imide bond in this domain is responsible for simultaneous existence of two distinct SH2 conformers. Prolyl isomerization directs ligand recognition: the trans conformer preferentially binds pY. The structure of the SH2/pY complex provides insight into the ligand specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged for optimal contacts with the pY+3 residue of the ligand. Analysis of (3h)J(NC') couplings arising from hydrogen bonds has revealed propagation of structural changes from the pY binding pocket to the CD loop containing conformationally heterogeneous proline as well as to the alphaB helix, on the opposite site of the domain. These findings offer a structural framework for understanding the roles of prolyl isomerization and pY binding in Itk regulation.     

The C. elegans Frizzled CFZ-2 is required for cell migration and interacts with multiple Wnt signaling pathways

Members of the Frizzled family of integral membrane proteins are implicated in many developmental events, including specifying cell fate, orienting cell and planar polarity, and directing cell migration. Frizzleds function as cell surface receptors for secreted Wnt proteins. We report here the isolation of a mutation in cfz-2, a Caenorhabditis elegans Frizzled gene. Mutation of cfz-2 causes defective cell migration, disorganization of head neurons, and can cause ectopic axon outgrowth. Analysis of mosaic animals shows that CFZ-2 functions cell nonautonomously, but does not rule out an autonomous role. CFZ-2 is expressed primarily in the anterior of embryos and in several cells in the head of adults. Our analysis of interactions between CFZ-2 and other Wnt pathways reveals that three Wnts, CWN-1, CWN-2 and EGL-20, and a Frizzled, MOM-5, function redundantly with one another and with CFZ-2 for specific cell migrations. In contrast, CWN-1, CWN-2, EGL-20, CFZ-2, and MOM-5 antagonize one another for other migrations. Therefore, CFZ-2 functions by collaborating with and/or antagonizing other Wnt signaling pathways to regulate specific cell migrations.     

Transmembrane domain is crucial to the subcellular localization and function of Myc target 1

Deregulation of c‐MYC occurs in a variety of human cancers. Overexpression of c‐MYC promotes cell growth, proliferation, apoptosis, transformation and genomic instability. MYC target 1 (MYCT1) is a direct target gene of c‐MYC, and its murine homologue MT‐MC1 recapitulated multiple c‐Myc‐related phenotypes. However, the molecular mechanism of MYCT1 remains unclear. Here, we identified the transmembrane (TM) domain of MYCT1, not the nuclear localization sequence, is indispensable to the vesicle‐associated localization of MYCT1 protein in the cytoplasmic membrane vesicle. Overexpression of MYCT1, not MYCT1 (ΔTM), decreased cell viability under serum deprivation and increased tumour cell migration ability. We further identified CKAP4 interacted with MYCT1 and contributed to the function of MYCT1. In addition, we found that a mutation, A88D, which is observed in patient sample, changed the localization, and abolished the effect on cell viability and cell migration, suggesting that the TM domain is critical to MYCT1.     

Electrostatic interactions in the reconstitution of an SH2 domain from constituent peptide fragments

Fragment complementation has been used to delineate the essential recognition elements for stable folding in Src homology 2 (SH2) domains by using NMR spectroscopy, alanine scanning, and surface plasmon resonance. The unfolded 9-kD and 5-kD peptide fragments formed by limited proteolytic digestion of the N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3'-kinase fold into an active native-like structure on interaction with one another. The corresponding 5-kD fragment of the homologous Src protein, however, was not capable of structurally complementing the p85 9-kD fragment, indicating that fragment complementation among these SH2 domains is sensitive to the sequence differences between the Src and p85 domains. Partial complementation and folding activity could be recovered with hybrid sequences of these SH2 domains. Complete alanine scanning of the 5-kD p85 fragment was used to identify the sequence recognition elements required for complex formation. The alanine substitutions in the p85 5-kD fragment that abolished binding affinity with the cognate 9-kD fragment correlate well with highly conserved residues among SH2 domains that are either integrally involved in core packing or found at the interface between fragments. Surprisingly, however, mutation of a nonconserved surface-exposed aspartic acid to alanine was found to have a significant effect on complementation. A single additional mutation of arginine to aspartic acid allowed for recovery of native structure and increased the thermal stability of the designed Src-p85 chimera by 18 degrees C. This modification appears to relieve an unfavorable surface electrostatic interaction, demonstrating the importance of surface charge interactions in protein stability.     

Cerebellar cortical lamination and foliation require cyclin A2

The mammalian genome encodes two A-type cyclins, which are considered potentially redundant yet essential regulators of the cell cycle. Here, we tested requirements for cyclin A1 and cyclin A2 function in cerebellar development. Compound conditional loss of cyclin A1/A2 in neural progenitors resulted in severe cerebellar hypoplasia, decreased proliferation of cerebellar granule neuron progenitors (CGNP), and Purkinje (PC) neuron dyslamination. Deletion of cyclin A2 alone showed an identical phenotype, demonstrating that cyclin A1 does not compensate for cyclin A2 loss in neural progenitors. Cyclin A2 loss lead to increased apoptosis at early embryonic time points but not at post-natal time points. In contrast, neural progenitors of the VZ/SVZ did not undergo increased apoptosis, indicating that VZ/SVZ-derived and rhombic lip-derived progenitor cells show differential requirements to cyclin A2. Conditional knockout of cyclin A2 or the SHH proliferative target Nmyc in CGNP also resulted in PC neuron dyslamination. Although cyclin E1 has been reported to compensate for cyclin A2 function in fibroblasts and is upregulated in cyclin A2 null cerebella, cyclin E1 expression was unable to compensate for loss-of cyclin A2 function.     

Overexpression of Kelch domain containing-2 (mKlhdc2) inhibits differentiation and directed migration of C2C12 myoblasts

Targeted migration of muscle precursor cells to the anlagen of limb muscles is a complex process, which is only partially understood. We have used Lbx1 mutant mice, which are unable to establish correct migration paths of muscle precursor cells into the limbs to identify new genes involved in the accurate placement of myogenic cells in developing muscles. We found that mKlhdc2 (Kelch domain containing-2), a novel member of the family of Kelch domain containing proteins, is significantly downregulated in Lbx1 homozygous mutant embryos. Functional characterization of mKlhdc2 by targeted overexpression in 10T1/2 fibroblasts and C2C12 muscle cells rendered these cells unable to respond to chemoattractants such as HGF. Furthermore, C2C12 myoblasts overexpressing mKlhdc2 display altered cellular morphology and are unable to differentiate into mature myotubes. Our results suggest that a tightly controlled expression of mKlhdc2 is essential for a faithful execution of the myogenic differentiation and migration program.     

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.     

The NC1/endostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance

Type XVIII collagen is a homotrimeric basement membrane molecule of unknown function, whose COOH-terminal NC1 domain contains endostatin (ES), a potent antiangiogenic agent. The Caenorhabditis elegans collagen XVIII homologue, cle-1, encodes three developmentally regulated protein isoforms expressed predominantly in neurons. The CLE-1 protein is found in low amounts in all basement membranes but accumulates at high levels in the nervous system. Deletion of the cle-1 NC1 domain results in viable fertile animals that display multiple cell migration and axon guidance defects. Particular defects can be rescued by ectopic expression of the NC1 domain, which is shown to be capable of forming trimers. In contrast, expression of monomeric ES does not rescue but dominantly causes cell and axon migration defects that phenocopy the NC1 deletion, suggesting that ES inhibits the promigratory activity of the NC1 domain. These results indicate that the cle-1 NC1/ES domain regulates cell and axon migrations in C. elegans.