Brain cDNA Research Articles

Alternative Splicing of the Last TKFC Intron Yields Transcripts Differentially Expressed in Human Tissues That Code In Vitro for a Protein Devoid of Triokinase and FMN Cyclase Activity.

The 18-exon human TKFC gene codes for dual-activity triokinase and FMN cyclase (TKFC) in an ORF, spanning from exon 2 to exon 18. In addition to TKFC-coding transcripts (classified as tkfc type by their intron-17 splice), databases contain evidence for alternative TKFC transcripts, but none of them has been expressed, studied, and reported in the literature. A novel full-ORF transcript was cloned from brain cDNA and sequenced (accession no. DQ344550). It results from an alternative 3' splice-site in intron 17. The cloned cDNA contains an ORF also spanning from exon 2 to exon 18 of the TKFC gene but with a 56-nt insertion between exons 17 and 18 (classified as tkfc_ins56 type). This insertion introduces an in-frame stop, and the resulting ORF codes for a shorter TKFC variant, which, after expression, is enzymatically inactive. TKFC intron-17 splicing was found to be differentially expressed in human tissues. In a multiple-tissue northern blot using oligonucleotide probes, the liver showed a strong expression of the tkfc-like splice of intron 17, and the heart preferentially expressed the tkfc_ins56-like splice. Through a comparison to global expression data from massive-expression studies of human tissues, it was inferred that the intestine preferentially expresses TKFC transcripts that contain neither of those splices. An analysis of transcript levels quantified by RNA-Seq in the GTEX database revealed an exception to this picture due to the occurrence of a non-coding short transcript with a tkfc-like splice. Altogether, the results support the occurrence of potentially relevant transcript variants of the TKFC gene, differentially expressed in human tissues. (This work is dedicated in memoriam to Professor Antonio Sillero, 1938-2024, for his lifelong mentoring and his pioneering work on triokinase).

Experimental and computational evidence that Calpain-10 binds to the carboxy terminus of NaV1.2 and NaV1.6

Voltage-gated sodium channels (NaV) are pivotal proteins responsible for initiating and transmitting action potentials. Emerging evidence suggests that proteolytic cleavage of sodium channels by calpains is pivotal in diverse physiological scenarios, including ischemia, brain injury, and neuropathic pain associated with diabetes. Despite this significance, the precise mechanism by which calpains recognize sodium channels, especially given the multiple calpain isoforms expressed in neurons, remains elusive. In this work, we show the interaction of Calpain-10 with NaV's C-terminus through a yeast 2-hybrid assay screening of a mouse brain cDNA library and in vitro by GST-pulldown. Later, we also obtained a structural and dynamic hypothesis of this interaction by modeling, docking, and molecular dynamics simulation. These results indicate that Calpain-10 interacts differentially with the C-terminus of NaV1.2 and NaV1.6. Calpain-10 interacts with NaV1.2 through domains III and T in a stable manner. In contrast, its interaction with NaV1.6 involves domains II and III, which could promote proteolysis through the Cys-catalytic site and C2 motifs.

Identification of binding partners that facilitate membrane-type 5 matrix metalloproteinase (MT5-MMP) processing of amyloid precursor protein.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of extracellular deposits of amyloid beta (Aβ) peptide. In addition to Aβ as the core component of the amyloid plaque, the amyloid precursor protein (APP) processing fragment Aβ was also found accumulated around the plaque. The APPη pathway, mainly mediated by membrane-type 5 matrix metalloproteinase (MT5-MMP), represents an important factor in AD pathogenesis. The proamyloidogenic features of MT5-MMP could result from interactions with APP when trafficking between organelles, so determination of the location within the cell of APPη cleavage and interacting proteins of MT5-MMP affecting this process will be of priority in understanding the role of MT5-MMP in AD. In the present study,MT5-MMP was found to be located in the nucleus, cytosol, and cytosolic subcellular granules of CHO cells that stably expressed wild-type human APP751. MT5-MMP fusion proteins were constructed that could localize enzyme production in the Golgi apparatus, endosome, ER, mitochondria, or plasma membrane. The fusion proteins significantly increased sAPPη when directed to the endosome, Golgi apparatus, plasma membrane, or mitochondria. Since the C-terminal region of MT5-MMP is responsible for its intracellular location and trafficking, this domain was used as the bait in a yeast two-hybrid screen to identify MT5-MMP protein partners in a human brain cDNA library. Identified binding partners included N4BP2L1, TMX3, EIG121, bridging Integrator 1(BIN1), RUFY4, HTRA1, and TMEM199. The binding of N4BP2L1, EIG121, BIN1, or TMX3 to MT5-MMP resulted in the most significant increase in sAPPη production. Thus, the action of MT5-MMP on APP occurs in multiple locations within the cell and is facilitated by site-specific binding partners.

A peptide rich in glycine-serine-alanine repeats ameliorates Alzheimer-type neurodegeneration.

Repeated amino acid sequences in proteins are widely found, and the glycine-serine-alanine repeat is an element with a general propensity to form β-sheet aggregates as found in key pathological factors, in several neurodegenerative diseases. Such properties of this repeat may guide development of disease-modifying therapies for neurodegenerative disease. However, details of its role and underlying mechanism(s) remain largely unknown. Actions of specific glycine-serine-alanine repeat peptides (SNPs), especially SNP-9, on Alzheimer's disease (AD)-like abnormalities were evaluated in transgenic mice and Caenorhabditis elegans, andin rat and cell models. Entry of SNPs into the brain, SNP activity in neuronal cells and peptide entry into cells were analysed in vivo and in vitro. Cell-free systems and the yeast two-hybrid system were also used to explore possible targets of SNP-9, and interactions of potential targets with SNP-9 were confirmed in cell-based systems. We first identified SNP-9 as a potent neuroprotective peptide with the activity to decrease oligomeric amyloid β (Aβ) via co-assembling with the toxic Aβ oligomer to form hetero-oligomers. Also, calcyclin-binding protein was found to act as a SNP-9-binding protein, by screening of a human brain cDNA library. Such binding showed that SNP-9 could regulate the abnormal hyperphosphorylation of tau via calcyclin-bindingprotein. Our study provides a foundation for development of SNPs, especially SNP-9, as potential therapeutic interventions for AD. We propose SNP-9 as a potential therapeutic agent for the treatment of AD.

Novel maternal autoantibodies in autism spectrum disorder: Implications for screening and diagnosis.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder for which early recognition is a major challenge. Autoantibodies against fetal brain antigens have been found in the blood of mothers of children with ASD (m-ASD) and can be transferred to the fetus where they can impact neurodevelopment by binding to fetal brain proteins. This study aims to identify novel maternal autoantibodies reactive against human fetal brain antigens, and explore their use as biomarkers for ASD screening and diagnosis. A custom-made human fetal brain cDNA phage display library was constructed, and screened for antibody reactivity in m-ASD samples from the Simons Simplex Collection (SSC) of the Simons Foundation Autism Research Initiative (SFARI). Antibody reactivity against 6 identified antigens was determined in plasma samples of 238 m-ASD and 90 mothers with typically developing children (m-TD). We identified antibodies to 6 novel University Hasselt (UH)-ASD antigens, including three novel m-ASD autoantigens, i.e., ribosomal protein L23 (RPL23), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calmodulin-regulated spectrin-associated protein 3 (CAMSAP3). Antibody reactivity against a panel of four of these targets was found in 16% of m-ASD samples, compared to 4% in m-TD samples (p = 0.0049). Maternal antibodies against 4 UH-ASD antigens could therefore provide a novel tool to support the diagnosis of ASD in a subset of individuals.

The Lambda Display Technology: A Useful Tool for the Identification of Ubiquitin-and Ubiquitin-Like-Binding Domains.

Ubiquitin-binding domains (UBDs) are modular units that mediate non-covalent recognition of ubiquitin modifications. They are found in ubiquitin (Ub)-binding proteins and recognize defined surface patches of a single Ub through typically weak interactions. Although more than 200 Ub-binding proteins have been identified to date, only 29 UBD types have been reported in the human proteome, suggesting that much remains to be learned about Ub recognition. Several methods, from bioinformatics to experimental, have successfully identified Ub-binding properties in several proteins. We here report the protocol to identify Ub-binding domains by panning a human brain cDNA library whose products are displayed on the surface of lambda capsid. In parallel, we carried out a panning experiment aimed at identifying domains interacting with the Ub-like NEDD8 (neural precursor cell-expressed developmentally downregulated), which is the Ub-like protein showing the closest sequence identity (58%) to Ub. This approach proved to be very effective for the discovery of the previously unidentified UBDs CUBAN and CoCUN, and it is in principle applicable to investigate the interaction network of any other Ub-like protein.

The aquaporin 8 (AQP8) membrane channel gene is present in the elasmobranch dogfish (Squalus acanthias) genome and is expressed in brain but not in gill, kidney or intestine.

The transport mechanisms for water, ammonia and urea in elasmobranch gill, kidney and gastrointestinal tract remain to be fully elucidated. Aquaporin 8 (AQP8) is a known water, ammonia and urea channel that is expressed in the kidney and respiratory and gastrointestinal tracts of mammals and teleost fish. However, at the initiation of this study in late 2019, there was no copy of an elasmobranch aquaporin 8 gene identified in the genebank even for closely related holocephalon species such as elephant fish (Callorhinchus milii) or for the elasmobranch little skate (Leucoraja erinacea). A transcriptomic study in spiny dogfish (Squalus acanthias) also failed to identify a copy. Hence this study has remedied this and identified the AQP8 cDNA sequence using degenerate PCR. Agarose electrophoresis of degenerate PCR reactions from dogfish tissues showed a strong band from brain cDNA and faint bands of a similar size in gill and liver. 5' and 3' RACE was used to complete the AQP8 cDNA sequence. Primers were then designed for further PCR reactions to determine the distribution of AQP8 mRNA expression in dogfish tissues. This showed that AQP8 is only expressed in dogfish brain and AQP8 therefore clearly can play no role in water, ammonia and urea transport in the gill, kidney or gastrointestinal tract. The role of AQP8 in dogfish brain remains to be determined.

COP1 Acts as a Ubiquitin Ligase for PCDH9 Ubiquitination and Degradation in Human Glioma.

Constitutive photomorphogenic 1 (COP1, also known as RFWD2), a ring-finger-type E3 ubiquitin ligase, has been reported to play a pivotal role in the regulation of cell growth, apoptosis, and DNA repair. Accumulating evidence has suggested that COP1 plays a role in tumorigenesis by triggering the ubiquitination and degradation of its substrates, but the potential mechanism remains unclear. In this study, COP1 was used as a bait in a yeast two-hybrid experiment to screen COP1-interacting proteins in a human brain cDNA library, and the results indicated that protocadherin 9 (PCDH9) was a potential binding protein of COP1. The interaction between and colocalization of COP1 and PCDH9 was further confirmed by coimmunoprecipitation (co-IP) assay and immunofluorescent staining. Subsequently, we demonstrated that COP1 acted as an E3 ligase to promote the ubiquitination and degradation of PCDH9 through the proteasome pathway in glioma cells. Furthermore, we identified that the type of COP1 mediated PCDH9 ubiquitination was Lys48-linked polyubiquitination. Finally, we found that the COP1 protein level was inversely correlated with the PCDH9 protein level in human glioma tissues. Taken together, our results suggest that COP1 is an E3 ubiquitin ligase for PCDH9 and reveal an important mechanism for PCDH9 regulation in human glioma.

Calsyntenin-3 interacts with the sodium-dependent vitamin C transporter-2 to regulate vitamin C uptake

Ascorbic acid (AA) uptake in neurons occurs via a Na+-dependent carrier-mediated process mediated by the sodium-dependent vitamin C transporter-2 (SVCT2). Relatively little information is available concerning the network of interacting proteins that support human (h)SVCT2 trafficking and cell surface expression in neuronal cells. Here we identified the synaptogenic adhesion protein, calsyntenin-3 (CLSTN3) as an hSVCT2 interacting protein from yeast two-hybrid (Y2H) screening of a human adult brain cDNA library. This interaction was confirmed by co-immunoprecipitation, mammalian two-hybrid (M2H), and co-localization in human cell lines. Co-expression of hCLSTN3 with hSVCT2 in SH-SY5Y cells led to a marked increase in AA uptake. Reciprocally, siRNA targeting hCLSTN3 inhibited AA uptake. In the J20 mouse model of Alzheimer's disease (AD), mouse (m)SVCT2 and mCLSTN3 expression levels in hippocampus were decreased. Similarly, expression levels of hSVCT2 and hCLSTN3 were markedly decreased in hippocampal samples from AD patients. These findings establish CLSTN3 as a novel hSVCT2 interactor in neuronal cells with potential pathophysiological significance.

APP Binds to the EGFR Ligands HB-EGF and EGF, Acting Synergistically with EGF to Promote ERK Signaling and Neuritogenesis.

The amyloid precursor protein (APP) is a transmembrane glycoprotein central to Alzheimer's disease (AD) with functions in brain development and plasticity, including in neurogenesis and neurite outgrowth. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are well-described neurotrophic and neuromodulator EGFR ligands, both implicated in neurological disorders, including AD. Pro-HB-EGF arose as a putative novel APP interactor in a human brain cDNA library yeast two-hybrid screen. Based on their structural and functional similarities, we first aimed to verify if APP could bind to (HB-)EGF proforms. Here, we show that APP interacts with these two EGFR ligands, and further characterized the effects of APP-EGF interaction in ERK activation and neuritogenesis. Yeast co-transformation and co-immunoprecipitation assays confirmed APP interaction with HB-EGF. Co-immunoprecipitation also revealed that APP binds to cellular pro-EGF. Overexpression of HB-EGF in HeLa cells, or exposure of SH-SY5Y cells to EGF, both resulted in increased APP protein levels. EGF and APP were observed to synergistically activate the ERK pathway, crucial for neuronal differentiation. Immunofluorescence analysis of cellular neuritogenesis in APP overexpression and EGF exposure conditions confirmed a synergistic effect in promoting the number and the mean length of neurite-like processes. Synergistic ERK activation and neuritogenic effects were completely blocked by the EGFR inhibitor PD 168393, implying APP/EGF-induced activation of EGFR as part of the mechanism. This work shows novel APP protein interactors and provides a major insight into the APP/EGF-driven mechanisms underlying neurite outgrowth and neuronal differentiation, with potential relevance for AD and for adult neuroregeneration.

The Human Isoform of RNA Polymerase II Subunit hRPB11bα Specifically Interacts with Transcription Factor ATF4.

Rpb11 subunit of RNA polymerase II of Eukaryotes is related to N-terminal domain of eubacterial α subunit and forms a complex with Rpb3 subunit analogous to prokaryotic α2 homodimer, which is involved in RNA polymerase assembly and promoter recognition. In humans, a POLR2J gene family has been identified that potentially encodes several hRPB11 proteins differing mainly in their short C-terminal regions. The functions of the different human specific isoforms are still mainly unknown. To further characterize the minor human specific isoform of RNA polymerase II subunit hRPB11bα, the only one from hRPB11 (POLR2J) homologues that can replace its yeast counterpart in vivo, we used it as bait in a yeast two-hybrid screening of a human fetal brain cDNA library. By this analysis and subsequent co-purification assay in vitro, we identified transcription factor ATF4 as a prominent partner of the minor RNA polymerase II (RNAP II) subunit hRPB11bα. We demonstrated that the hRPB11bα interacts with leucine b-Zip domain located on the C-terminal part of ATF4. Overexpression of ATF4 activated the reporter more than 10-fold whereas co-transfection of hRPB11bα resulted in a 2.5-fold enhancement of ATF4 activation. Our data indicate that the mode of interaction of human RNAP II main (containing major for of hRPB11 subunit) and minor (containing hRPB11bα isoform of POLR2J subunit) transcription enzymes with ATF4 is certainly different in the two complexes involving hRPB3–ATF4 (not hRPB11a–ATF4) and hRpb11bα–ATF4 platforms in the first and the second case, respectively. The interaction of hRPB11bα and ATF4 appears to be necessary for the activation of RNA polymerase II containing the minor isoform of the hRPB11 subunit (POLR2J) on gene promoters regulated by this transcription factor. ATF4 activates transcription by directly contacting RNA polymerase II in the region of the heterodimer of α-like subunits (Rpb3–Rpb11) without involving a Mediator, which provides fast and highly effective activation of transcription of the desired genes. In RNA polymerase II of Homo sapiens that contains plural isoforms of the subunit hRPB11 (POLR2J), the strength of the hRPB11–ATF4 interaction appeared to be isoform-specific, providing the first functional distinction between the previously discovered human forms of the Rpb11 subunit.

Identification of novel non-myelin biomarkers in multiple sclerosis using an improved phage-display approach

Although the etiology of multiple sclerosis is not yet understood, it is accepted that its pathogenesis involves both autoimmune and neurodegenerative processes, in which the role of autoreactive T-cells has been elucidated. Instead, the contribution of humoral response is still unclear, even if the presence of intrathecal antibodies and B-cells follicle-like structures in meninges of patients has been demonstrated. Several myelin and non-myelin antigens have been identified, but none has been validated as humoral biomarker. In particular autoantibodies against myelin proteins have been found also in healthy individuals, whereas non-myelin antigens have been implicated in neurodegenerative phase of the disease. To provide further putative autoantigens of multiple sclerosis, we investigated the antigen specificity of immunoglobulins present both in sera and in cerebrospinal fluid of patients using phage display technology in a new improved format. A human brain cDNA phage display library was constructed and enriched for open-read-frame fragments. This library was selected against pooled and purified immunoglobulins from cerebrospinal fluid and sera of multiple sclerosis patients. The antigen library was also screened against an antibody scFv library obtained from RNA of B cells purified from the cerebrospinal fluid of two relapsing remitting patients. From all biopanning a complex of 14 antigens were identified; in particular, one of these antigens, corresponding to DDX24 protein, was present in all selections. The ability of more frequently isolated antigens to discriminate between sera from patients with multiple sclerosis or other neurological diseases was investigated. The more promising novel candidate autoantigens were DDX24 and TCERG1. Both are implicated in RNA modification and regulation which can be altered in neurodegenerative processes. Therefore, we propose that they could be a marker of a particular disease activity state.

Hsp40 Protein DNAJB6 Interacts with Viral NS3 and Inhibits the Replication of the Japanese Encephalitis Virus.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus prevalent in east and southeast Asia, the Western Pacific, and northern Australia. Since viruses are obligatory intracellular pathogens, the dynamic processes of viral entry, replication, and assembly are dependent on numerous host-pathogen interactions. Efforts to identify JEV-interacting host factors are ongoing because their identification and characterization remain incomplete. Three enzymatic activities of flavivirus non-structural protein 3 (NS3), including serine protease, RNA helicase, and triphosphatase, play major roles in the flaviviruses lifecycle. To identify cellular factors that interact with NS3, we screened a human brain cDNA library using a yeast two-hybrid assay, and identified eight proteins that putatively interact with NS3: COPS5, FBLN5, PPP2CB, CRBN, DNAJB6, UBE2N, ZNF350, and GPR137B. We demonstrated that the DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6) colocalizes and interacts with NS3, and has a negative regulatory function in JEV replication. We also show that loss of DNAJB6 function results in significantly increased viral replication, but does not affect viral binding or internalization. Moreover, the time-course of DNAJB6 disruption during JEV infection varies in a viral load-dependent manner, suggesting that JEV targets this host chaperone protein for viral benefit. Deciphering the modes of NS3-interacting host proteins functions in virion production will shed light on JEV pathogenic mechanisms and may also reveal new avenues for antiviral therapeutics.

Surface expression of TTYH2 is attenuated by direct interaction with β-COP

TTYH2 is a calcium-activated, inwardly rectifying anion channel that has been shown to be related to renal cancer and colon cancer. Based on the topological prediction, TTYH2 protein has five transmembrane domains with the extracellular N-terminus and the cytoplasmic C-terminus. In the present study, we identified a vesicle transport protein, β-COP, as a novel specific binding partner of TTYH2 by yeast two-hybrid screening using a human brain cDNA library with the C-terminal region of TTYH2 (TTYH2-C) as a bait. Using in vitro and in vivo binding assays, we confirmed the protein-protein interactions between TTYH2 and β-COP. We also found that the surface expression and activity of TTYH2 were decreased by co-expression with β-COP in the heterologous expression system. In addition, β-COP associated with TTYH2 in a native condition at a human colon cancer cell line, LoVo cells. The over-expression of β-COP in the LoVo cells led to a dramatic decrease in the surface expression and activity of endogenous TTYH2. Collectively, these data suggested that β-COP plays a critical role in the trafficking of the TTYH2 channel to the plasma membrane.

Biosynthetic Short Neuropeptides: A Rational Theory Based on Experimental Results for the Missing Pain-Relief Opioid Endomorphin Precursor Gene.

Endomorphins are neuropeptides that bind strongly to μ-opioid receptors and are considered to play important roles in pain modulation and other biological functions. Two endomorphins have been identified, to date, endomorphine-1 and -2; both are tetrapeptides and differ by only a single amino acid in the third position. Both peptides were isolated from bovine brains; however, their precursor genes have not been identified. In this study, a nucleotide sequence corresponding to the endomorphin-1 peptide in an expressed sequence tag database has been found and a preproendomorphin-like precursor peptide from human brain complementary DNA (cDNA) has been cloned. The cDNA consists of nucleotide sequences of two already annotated predicted genes, and the putative peptide differs by one amino acid from the isolated endomorphin peptides. It is proposed herein that there is the possibility of unknown short proteins or peptide precursors being missed by automated gene prediction programs based on similarities of known protein sequences. A novel concept of how to produce endomorphins from a similar peptide is described. The oxidatively modified base might provide a clue for understanding discrepancies between nucleotide sequences on the genome and those on cDNAs.

Analysis of Uncharacterized mKiaa1211 Expression during Mouse Development and Cardiovascular Morphogenesis.

Mammalian Kiaa1211 and Kiaa1211-like are a homologous pair of uncharacterized, highly conserved genes cloned from fetal and adult brain cDNA libraries. Herein we map the in utero spatiotemporal expression of mKiaa1211 and mKiaa1211L mRNA and their expression patterns in postnatal testis, skin, gastrointestinal, and adipose progenitor tissues. Significantly, mKiaa1211 is present throughout the early stages of mouse heart development, particularly in the second heart field (SHF) lineage as it differentiates from mesenchymal cells into cardiomyocytes. We also show that mKiaa1211 is expressed within several early neuronal tissues destined to give rise to central, peripheral, and sympathetic nervous system structures. Expression profiling revealed that the paralog mKiaa1211L is not expressed during the normal developmental process and that mKiaa1211 expression was noticeably absent from most adult terminally differentiated tissues. Finally, we confirm that a previously uncharacterized CRISPR/CAS-generated mKiaa1211 mouse mutant allele is hypomorphic.

Implications of the mitochondrial interactome of mammalian thioredoxin 2 for normal cellular function and disease

Multiple thioredoxin isoforms exist in all living cells. To explore the possible functions of mammalian mitochondrial thioredoxin 2 (Trx2), an interactome of mouse Trx2 was initially created using (i) a monothiol mouse Trx2 species for capturing protein partners from different organs and (ii) yeast two hybrid screens on human liver and rat brain cDNA libraries. The resulting interactome consisted of 195 proteins (Trx2 included) plus the mitochondrial 16S RNA. 48 of these proteins were classified as mitochondrial (MitoCarta2.0 human inventory). In a second step, the mouse interactome was combined with the current four-membered mitochondrial sub-network of human Trx2 (BioGRID) to give a 53-membered human Trx2 mitochondrial interactome (52 interactor proteins plus the mitochondrial 16S RNA). Although thioredoxins are thiol-employing disulfide oxidoreductases, approximately half of the detected interactions were not due to covalent disulfide bonds. This finding reinstates the extended role of thioredoxins as moderators of protein function by specific non-covalent, protein-protein interactions. Analysis of the mitochondrial interactome suggested that human Trx2 was involved potentially in mitochondrial integrity, formation of iron sulfur clusters, detoxification of aldehydes, mitoribosome assembly and protein synthesis, protein folding, ADP ribosylation, amino acid and lipid metabolism, glycolysis, the TCA cycle and the electron transport chain. The oxidoreductase functions of Trx2 were verified by its detected interactions with mitochondrial peroxiredoxins and methionine sulfoxide reductase. Parkinson's disease, triosephosphate isomerase deficiency, combined oxidative phosphorylation deficiency, and lactate dehydrogenase b deficiency are some of the diseases where the proposed mitochondrial network of Trx2 may be implicated.

Identification of FAM96B as a novel selenoprotein W binding partner in the brain

Selenoprotien W (SelW) plays a key role in brain development, although the exact biological function and mechanisms remain unclear. We performed a yeast two-hybrid screen on a human fetal brain cDNA library and identified FAM96B as a novel binding partner of SelW. FRET analyses confirmed the interaction between SelW' and FAM96B. The mutated SelW' construct was cloned and overexpressed in E. coli, and a pull-down assay verified a direct interaction between SelW' and FAM96B. Finally, Co-Immunoprecipitation on murine brain tissue proteins demonstrated an endogenous interaction between the two proteins in the brain. Taken together, our findings prove a direct interaction between SelW and FAM96B, which may provide new insights into the role of SelW in brain development and neurodegenerative diseases.

Identification of C-Terminal Binding Protein 1 as a Novel NMDA Receptor Interactor

A new N-methyl D aspartate neurotransmitter receptor interacting protein has been identified by yeast two-hybrid screening of a mouse brain cDNA library. C-terminal binding protein 1 (CtBP1) was shown to associate with the intracellular C-terminal regions of the N-methyl D aspartate receptor subunits GluN2A and GluN2D but not with GluN1-1a cytoplasmic C-terminal region. In yeast mating assays using a series of GluN2A C-terminal truncations, it was demonstrated that the CtBP1 binding domain was localized to GluN2A 1157–1382. The GluN2A binding domain was identified to lie within the CtBP1 161–224 region. CtBP1 co-immunoprecipitated with assembled GluN1/GluN2A receptors expressed in mammalian cells and also, in detergent extracts of adult mouse brain. Co-expression of CtBP1 with GluN1/GluN2A resulted in a significant decrease in receptor cell surface expression. The family of C-terminal binding proteins function primarily as transcriptional co-repressors. However, they are also known to modulate intracellular membrane trafficking mechanisms. Thus the results reported herein describe a putative role for CtBP1 in the regulation of cell surface N-methyl D aspartate receptor expression.

Pallidin is a novel interacting protein for cytohesin-2 and regulates the early endosomal pathway and dendritic formation in neurons.

Cytohesin-2 is a member of the guanine nucleotide exchange factors for ADP ribosylation factor 1 (Arf1) and Arf6, which are small GTPases that regulate membrane traffic and actin dynamics. In this study, we first demonstrated that cytohesin-2 localized to the plasma membrane and vesicles in various subcellular compartment in hippocampal neurons by immunoelectron microscopy. Next, to understand the molecular network of cytohesin-2 in neurons, we conducted yeast two-hybrid screening of brain cDNA libraries using cytohesin-2 as bait and isolated pallidin, a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) involved in endosomal trafficking. Pallidin interacted specifically with cytohesin-2 among cytohesin family members. Glutathione S-transferase pull-down and immunoprecipitation assays further confirmed the formation of a protein complex between cytohesin-2 and pallidin. Immunofluorescence demonstrated that cytohesin-2 and pallidin partially colocalized in various subsets of endosomes immunopositive for EEA1, syntaxin 12, and LAMP2 in hippocampal neurons. Knockdown of pallidin or cytohesin-2 reduced cytoplasmic EEA1-positive early endosomes. Furthermore, knockdown of pallidin increased the total dendritic length of cultured hippocampal neurons, which was rescued by co-expression of wild-type pallidin but not a mutant lacking the ability to interact with cytohesin-2. In contrast, knockdown of cytohesin-2 had the opposite effect on total dendritic length. The present results suggested that the interaction between pallidin and cytohesin-2 may participate in various neuronal functions such as endosomal trafficking and dendritic formation in hippocampal neurons. Cover Image for this issue: doi: 10.1111/jnc.14197.