The Fusion of CLEC12A and MIR223HG Arises from a trans-Splicing Event in Normal and Transformed Human Cells.

Chimeric RNAs are often associated with chromosomal rearrangements in cancer. In addition, they are also widely detected in normal tissues, contributing to transcriptomic complexity. Despite their prevalence, little is known about the characteristics and functions of chimeric RNAs. Here, we examine the genetic structure and biological roles of CLEC12A-MIR223HG, a novel chimeric transcript produced by the fusion of the cell surface receptor CLEC12A and the miRNA-223 host gene (MIR223HG), first identified in chronic myeloid leukemia (CML) patients. Surprisingly, we observed that CLEC12A-MIR223HG is not just expressed in CML, but also in a variety of normal tissues and cell lines. CLEC12A-MIR223HG expression is elevated in pro-monocytic cells resistant to chemotherapy and during monocyte-to-macrophage differentiation. We observed that CLEC12A-MIR223HG is a product of trans-splicing rather than a chromosomal rearrangement and that transcriptional activation of CLEC12A with the CRISPR/Cas9 Synergistic Activation Mediator (SAM) system increases CLEC12A-MIR223HG expression. CLEC12A-MIR223HG translates into a chimeric protein, which largely resembles CLEC12A but harbours an altered C-type lectin domain altering key disulphide bonds. These alterations result in differences in post-translational modifications, cellular localization, and protein-protein interactions. Taken together, our observations support a possible involvement of CLEC12A-MIR223HG in the regulation of CLEC12A function. Our workflow also serves as a template to study other uncharacterized chimeric RNAs.

Plasticity of the lettuce infectious yellows virus minor coat protein (CPm) in mediating the foregut retention and transmission of a chimeric CPm mutant by whitefly vectors.

Transmission of the crinivirus, lettuce infectious yellows virus (LIYV), is determined by a minor coat protein (CPm)-mediated virion retention mechanism located in the foregut of its whitefly vector. To better understand the functions of LIYV CPm, chimeric CPm mutants engineered with different lengths of the LIYV CPm amino acid sequence and that of the crinivirus, lettuce chlorosis virus (LCV), were constructed based on bioinformatics and sequence alignment data. The 485 amino acid-long chimeric CPm of LIYV mutant, CPmP-1, contains 60 % (from position 3 to 294) of LCV CPm amino acids. The chimeric CPm of mutants CPmP-2, CPmP-3 and CPmP-4 contains 46 (position 3 to 208), 51 (position 3 to 238) and 41 % (position 261 to 442) of LCV CPm amino acids, respectively. All four mutants moved systemically, expressed the chimeric CPm and formed virus particles. However, following acquisition feeding of the virus preparations, only CPmP-1 was retained in the foreguts of a significant number of vectors and transmitted. In immuno-gold labelling transmission electron microscopy (IGL-TEM) analysis, CPmP-1 particles were distinctly labelled by antibodies directed against the LCV but not LIYV CPm. In contrast, CPmP-4 particles were not labelled by antibodies directed against the LCV or LIYV CPm, while CPmP-2 and -3 particles were weakly labelled by anti-LIYV CPm but not anti-LCV CPm antibodies. The unique antibody recognition and binding pattern of CPmP-1 was also displayed in the foreguts of whitefly vectors that fed on CPmP-1 virions. These results are consistent with the hypothesis that the chimeric CPm of CPmP-1 is incorporated into functional virions, with the LCV CPm region being potentially exposed on the surface and accessible to anti-LCV CPm antibodies.

Systematic mining of fungal chimeric terpene synthases using an efficient precursor-providing yeast chassis.

Chimeric terpene synthases, which consist of C-terminal prenyltransferase (PT) and N-terminal class I terpene synthase (TS) domains (termed PTTSs here), is unique to fungi and produces structurally diverse di- and sesterterpenes. Prior to this study, 20 PTTSs had been functionally characterized. Our understanding of the origin and functional evolution of PTTS genes is limited. Our systematic search of sequenced fungal genomes among diverse taxa revealed that PTTS genes were restricted to Dikarya. Phylogenetic findings indicated different potential models of the origin and evolution of PTTS genes. One was that PTTS genes originated in the common Dikarya ancestor and then underwent frequent gene loss among various subsequent lineages. To understand their functional evolution, we selected 74 PTTS genes for biochemical characterization in an efficient precursor-providing yeast system employing chassis-based, robot-assisted, high-throughput automatic assembly. We found 34 PTTS genes that encoded active enzymes and collectively produced 24 di- and sesterterpenes. About half of these di- and sesterterpenes were also the products of the 20 known PTTSs, indicating functional conservation, whereas the PTTS products included the previously unknown sesterterpenes, sesterevisene (1), and sesterorbiculene (2), suggesting that a diversity of PTTS products awaits discovery. Separating functional PTTSs into two monophyletic groups implied that an early gene duplication event occurred during the evolution of the PTTS family followed by functional divergence with the characteristics of distinct cyclization mechanisms.

Aberrant splicing in neuroblastoma generates RNA-fusion transcripts and provides vulnerability to spliceosome inhibitors.

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.

Point mutation in the stop codon of MAV_RS14660 increases the growth rate of Mycobacterium avium subspecies hominissuis.

Mycobacterium avium subspecies hominissuis (MAH) is a pathogen that causes various non-tuberculous mycobacterial diseases in humans and animals worldwide. Among the genus, MAH is characterized by relatively slow growth. Here, we isolated a rapidly growing variant of the MAH 104 strain. The variant strain (named N104) exhibited an enhanced growth rate and higher motility compared to the parent MAH 104 strain (P104). Whole-genome sequencing analysis of N104 revealed the loss of the stop codon of MAV_RS14660 due to a single nucleotide replacement, resulting in the substitution of the codon for tryptophan. Notably, exclusion of the stop codon ligated the open reading frames and caused the fusion of two adjacent proteins. A revertant parent strain, in which a mutation was introduced to restore the stop codon, revealed that elimination of the stop codon in MAV_RS14660 was responsible for the N104 phenotype. Furthermore, we analysed the phenotypes of the parent and mutated strains by determining the functions of the MAV_RS14660 and MAV_RS14655 coding regions flanking the stop codon. The mutant strains, expected to express a fusion protein, exhibited increased resistance to antimicrobial drugs and exogenous copper toxicity compared to that of the parent strains. These findings suggest that the fusion of the MAV_RS14660- and MAV_RS14655-encoding regions in the mutant N104 strain could be related to the modified functions of these intrinsic proteins.

Activated HoxB4-induced hematopoietic stem cells from murine pluripotent stem cells via long-term programming.

Hematopoietic stem cells (HSCs) are multipotent cells that form the entire blood system and have the potential to cure several pathogenic conditions directly or indirectly arising from defects within the HSC compartment. Pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs) can give rise to all embryonic cell types; however, efficient in vitro differentiation of HSCs from PSCs remains challenging. HoxB4 is a key regulator orchestrating the differentiation of PSCs into all cells types across the mesodermal lineage, including HSCs. Moreover, the ectopic expression of HoxB4 enhances the in vitro generation and expansion of HSCs. However, several aspects of HoxB4 biology including its regulatory functions are not fully understood. Here, we describe the role of HoxB4 in indirectly inhibiting the emergence of mature CD45+ HSCs from iPSCs in vitro. Forced activation of HoxB4 permitted long-term maintenance of functional hematopoietic stem and progenitor cells (HSPCs), which efficiently reconstituted hematopoiesis upon transplantation. Our method enables an easy and scalable in vitro platform for the generation of HSCs from iPSCs, which will ultimately lead to a better understanding of HSC biology and facilitate preparation of the roadma for producing an unrestricted supply of HSCs for several curative therapies.

Cell Fusion Induced by a Fusion-Active Form of Human Cytomegalovirus Glycoprotein B (gB) Is Inhibited by Antibodies Directed at Antigenic Domain 5 in the Ectodomain of gB.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected in utero Virus-neutralizing antibodies defined in vitro have been proposed to confer protection against HCMV infection, and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is nonfusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to requirements of related class III fusion proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) or baculovirus gp64. To explore requirements for gB's fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus-neutralizing gB-specific monoclonal antibodies (MAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing MAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB MAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection.IMPORTANCE HCMV is a major global health concern, and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug-resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and, hence, is often included as a component of intervention strategies. By generation of fusion-active gB chimeras, we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB's fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.

Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection.

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.

CCL20 is a novel ligand for the scavenging atypical chemokine receptor 4.

The chemokine CCL20 is broadly produced by endothelial cells in the liver, the lung, in lymph nodes and mucosal lymphoid tissues, and recruits CCR6 expressing leukocytes, particularly dendritic cells, mature B cells, and subpopulations of T cells. How CCL20 is systemically scavenged is currently unknown. Here, we identify that fluorescently labeled human and mouse CCL20 are efficiently taken-up by the atypical chemokine receptor ACKR4. CCL20 shares ACKR4 with the homeostatic chemokines CCL19, CCL21, and CCL25, although with a lower affinity. We demonstrate that all 4 human chemokines recruit ß-arrestin1 and ß-arrestin2 to human ACKR4. Similarly, mouse CCL19, CCL21, and CCL25 equally activate the human receptor. Interestingly, at the same chemokine concentration, mouse CCL20 did not recruit ß-arrestins to human ACKR4. Further cross-species analysis suggests that human ACKR4 preferentially takes-up human CCL20, whereas mouse ACKR4 similarly internalizes mouse and human CCL20. Furthermore, we engineered a fluorescently labeled chimeric chemokine consisting of the N-terminus of mouse CCL25 and the body of mouse CCL19, termed CCL25_19, which interacts with and is taken-up by human and mouse ACKR4.

Engineering chimeric diterpene synthases and isoprenoid biosynthetic pathways enables high-level production of miltiradiene in yeast.

Miltiradiene is a key intermediate in the biosynthesis of many important natural diterpene compounds with significant pharmacological activity, including triptolide, tanshinones, carnosic acid and carnosol. Sufficient accumulation of miltiradiene is vital for the production of these medicinal compounds. In this study, comprehensive engineering strategies were applied to construct a high-yielding miltiradiene producing yeast strain. First, a chassis strain that can accumulate 2.1 g L-1 geranylgeraniol was constructed. Then, diterpene synthases from various species were evaluated for their ability to produce miltiradiene, and a chimeric miltiradiene synthase, consisting of class II diterpene synthase (di-TPS) CfTPS1 from Coleus forskohlii (Plectranthus barbatus) and class I di-TPS SmKSL1 from Salvia miltiorrhiza showed the highest efficiency in the conversion of GGPP to miltiradiene in yeast. Moreover, the miltiradiene yield was further improved by protein modification, which resulted in a final yield of 550.7 mg L-1 in shake flasks and 3.5 g L-1 in a 5-L bioreactor. This work offers an efficient and green process for the production of the important intermediate miltiradiene, and lays a foundation for further pathway reconstruction and the biotechnological production of valuable natural diterpenes.

Interdomain communication suppressing high intrinsic ATPase activity of Sse1 is essential for its co-disaggregase activity with Ssa1.

In eukaryotes, Hsp110s are unambiguous cognates of the Hsp70 chaperones, in primary sequence, domain organization, and structure. Hsp110s function as nucleotide exchange factors (NEFs) for the Hsp70s although their apparent loss of Hsp70-like chaperone activity, nature of interdomain communication, and breadth of domain functions are still puzzling. Here, by combining single-molecule FRET, small angle X-ray scattering measurements (SAXS), and MD simulation, we show that yeast Hsp110, Sse1 lacks canonical Hsp70-like interdomain allostery. However, the protein exhibits unique noncanonical conformational changes within its domains. Sse1 maintains an open-lid substrate-binding domain (SBD) in close contact with its nucleotide-binding domain (NBD), irrespective of its ATP hydrolysis status. To further appreciate such ATP-hydrolysis-independent exhaustive interaction between two domains of Hsp110s, NBD-SBD chimera was constructed between Hsp110 (Sse1) and Hsp70 (Ssa1). In Sse1/Ssa1 chimera, we observed undocking of two domains leading to complete loss of NEF activity of Sse1. Interestingly, chimeric proteins exhibited significantly enhanced ATPase rate of Sse1-NBD compared to wild-type protein, implying that intrinsic ATPase activity of the protein remains mostly repressed. Apart from repressing the high ATPase activity of its NBD, interactions between two domains confer thermal stability to Sse1 and play critical role in the (co)chaperoning function of Sse1 in Ssa1-mediated disaggregation activity. Altogether, Sse1 exhibits a unique interdomain interaction, which is essential for its NEF activity, suppression of high intrinsic ATPase activity, co-chaperoning activity in disaggregase machinery, and stability of the protein.

The hybrid protein interactome contributes to rice heterosis as epistatic effects.

Heterosis is the phenomenon in which hybrid progeny exhibits superior traits in comparison with those of their parents. Genomic variations between the two parental genomes may generate epistasis interactions, which is one of the genetic hypotheses explaining heterosis. We postulate that protein-protein interactions specific to F1 hybrids (F1 -specific PPIs) may occur when two parental genomes combine, as the proteome of each parent may supply novel interacting partners. To test our assumption, an inter-subspecies hybrid interactome was simulated by in silico PPI prediction between rice japonica (cultivar Nipponbare) and indica (cultivar 9311). Four-thousand, six-hundred and twelve F1 -specific PPIs accounting for 20.5% of total PPIs in the hybrid interactome were found. Genes participating in F1 -specific PPIs tend to encode metabolic enzymes and are generally localized in genomic regions harboring metabolic gene clusters. To test the genetic effect of F1 -specific PPIs in heterosis, genomic selection analysis was performed for trait prediction with additive, dominant and epistatic effects separately considered in the model. We found that the removal of single nucleotide polymorphisms associated with F1 -specific PPIs reduced prediction accuracy when epistatic effects were considered in the model, but no significant changes were observed when additive or dominant effects were considered. In summary, genomic divergence widely dispersed between japonica and indica rice may generate F1 -specific PPIs, part of which may accumulatively contribute to heterosis according to our computational analysis. These candidate F1 -specific PPIs, especially for those involved in metabolic biosynthesis pathways, are worthy of experimental validation when large-scale protein interactome datasets are generated in hybrid rice in the future.

Differences in the acidic sensitivity of transient receptor potential vanilloid 1 (TRPV1) between chickens and mice.

Chickens, one of the most important industrial animals, are a biological animal model. Here we focused on the transient receptor potential vanilloid 1 (TRPV1) to understand the pain system for acidic stimuli in chickens compared with mice. By using a whole-cell patch clamp system, we confirmed that acidic stimuli activate both chicken TRPV1 (cTRPV1) and mouse TRPV1 (mTRPV1), but the peak current of cTRPV1 is lower than that of mTRPV1, and it is difficult to desensitize cTRPV1 with an acidic stimulus compared to mTRPV1. Since the C-terminal of the calmodulin (CaM) binding site in TRPV1 was reported as one of the important structures for TRPV1 desensitization, we made chimeric cTRPV1 in which the CaM binding site of chicken is changed to that of mouse (cTRPV1-mCaM). We also compared the acidic responses of native chicken dorsal root ganglion (DRG) cells with that of mouse DRG cells. The TRPV1-mCaM results showed that the desensitization of mutant cTRPV1 was similar to that of mTRPV1, and that the basal activities of mutant cTRPV1 were significantly higher than those of cTRPV1. It was also difficult to desensitize the chicken DRG cells with an acidic stimulus, unlike the mouse DRG cells. These results suggest that there are differences in the pain transduction systems for acidic stimuli between chickens and mice that are caused by the dysfunction of the C-terminal CaM biding site of cTRPV1. These results imply that chickens repeatedly feel weak pain from an acidic stimulus, without desensitization.

Bioinformatic and Functional Evaluation of Actinobacterial Piperazate Metabolism.

Piperazate (Piz) is a nonproteinogenic amino acid noted for its unusual N-N bond motif. Piz is a proline mimic that imparts conformational rigidity to peptides. Consequently, piperazyl molecules are often bioactive and desirable for therapeutic exploration. The in vitro characterization of Kutzneria enzymes KtzI and KtzT recently led to a biosynthetic pathway for Piz. However, Piz anabolism in vivo has remained completely uncharacterized. Herein, we describe the systematic interrogation of actinobacterial Piz metabolism using a combination of bioinformatics, genetics, and select biochemistry. Following studies in Streptomyces flaveolus, Streptomyces lividans, and several environmental Streptomyces isolates, our data suggest that KtzI-type enzymes are conditionally dispensable for Piz production. We also demonstrate the feasibility of Piz monomer production using engineered actinobacteria for the first time. Finally, we show that some actinobacteria employ fused KtzI-KtzT chimeric enzymes to produce Piz. Our findings have implications for future piperazyl drug discovery, pathway engineering, and fine chemical bioproduction.

FusionGDB: fusion gene annotation DataBase.

Gene fusion is one of the hallmarks of cancer genome via chromosomal rearrangement initiated by DNA double-strand breakage. To date, many fusion genes (FGs) have been established as important biomarkers and therapeutic targets in multiple cancer types. To better understand the function of FGs in cancer types and to promote the discovery of clinically relevant FGs, we built FusionGDB (Fusion Gene annotation DataBase) available at https://ccsm.uth.edu/FusionGDB. We collected 48 117 FGs across pan-cancer from three representative fusion gene resources: the improved database of chimeric transcripts and RNA-seq data (ChiTaRS 3.1), an integrative resource for cancer-associated transcript fusions (TumorFusions), and The Cancer Genome Atlas (TCGA) fusions by Gao et al. For these ∼48K FGs, we performed functional annotations including gene assessment across pan-cancer fusion genes, open reading frame (ORF) assignment, and retention search of 39 protein features based on gene structures of multiple isoforms with different breakpoints. We also provided the fusion transcript and amino acid sequences according to multiple breakpoints and transcript isoforms. Our analyses identified 331, 303 and 667 in-frame FGs with retaining kinase, DNA-binding, and epigenetic factor domains, respectively, as well as 976 FGs lost protein-protein interaction. FusionGDB provides six categories of annotations: FusionGeneSummary, FusionProtFeature, FusionGeneSequence, FusionGenePPI, RelatedDrug and RelatedDisease.

Identification of differentially expressed genes and typical fusion genes associated with three subtypes of breast cancer.

BACKGROUND: This study aimed to identify the differentially expressed genes (DEGs) and the typical fusion genes in different types of breast cancers using RNA-seq. METHODS: GSE52643 was downloaded from Gene Expression Omnibus, which included 1 normal sample (MCF10A) and 7 breast cancer samples (BT-474, BT-20, MCF7, MDA-MB-231, MDA-MB-468, T47D, and ZR-75-1). The transcript abundance and the DEGs screening were performed by Cufflinks. The functional and pathway enrichment was analyzed by Gostats. SnowShoes-FTD was applied to identify the fusion genes. RESULTS: We screened 430, 445, 397, 417, 369, 557, and 375 DEGs in BT-474, BT-20, MCF7, DA-MB-231, MDA-MB-468, T47D, and ZR-75-1, respectively, compared with MCF10A. DEGs in each comparison group (such as CD40 and CDH1) were significantly enriched in the functions of cell adhesion and extracellular matrix organization and pathways of CAMs and ECM receptor interaction. UCP2 was a common DEG in the 7 comparison groups. SFRP1 and MMP7 were significantly enriched in wnt/-catenin signaling pathway in MDA-MB-231. FAS was significantly enriched in autoimmune thyroid disease pathway in BT-474. Besides, we screened 96 fusion genes, such as ESR1-C6orf97 in ZR-75-1, COBRA1-C9orf167 in BT-20, and VAPB-IKZF3 and ACACA-STAC2 in BT-474. CONCLUSIONS: The DEGs such as SFRP1, MMP7, CDH1, FAS, and UCP2 might be the potential biomarkers in breast cancer. Furthermore, some pivotal fusion genes like ESR1-C6orf97 with COBRA1-C9orf167 and VAPB-IKZF3 with ACACA-STAC2 were found in Luminal A and Luminal B breast cancer, respectively.

Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing.

The development of robust, versatile and accurate toolsets is critical to facilitate therapeutic genome editing applications. Here we establish RNA-programmable Cas9-Cas9 chimeras, in single- and dual-nuclease formats, as versatile genome engineering systems. In both of these formats, Cas9-Cas9 fusions display an expanded targeting repertoire and achieve highly specific genome editing. Dual-nuclease Cas9-Cas9 chimeras have distinct advantages over monomeric Cas9s including higher target site activity and the generation of predictable precise deletion products between their target sites. At a therapeutically relevant site within the BCL11A erythroid enhancer, Cas9-Cas9 nucleases produced precise deletions that comprised up to 97% of all sequence alterations. Thus Cas9-Cas9 chimeras represent an important tool that could be particularly valuable for therapeutic genome editing applications where a precise cleavage position and defined sequence end products are desirable.

Signal peptide represses GluK1 surface and synaptic trafficking through binding to amino-terminal domain.

Kainate-type glutamate receptors play critical roles in excitatory synaptic transmission and synaptic plasticity in the brain. GluK1 and GluK2 possess fundamentally different capabilities in surface trafficking as well as synaptic targeting in hippocampal CA1 neurons. Here we find that the excitatory postsynaptic currents (EPSCs) are significantly increased by the chimeric GluK1(SPGluK2) receptor, in which the signal peptide of GluK1 is replaced with that of GluK2. Coexpression of GluK1 signal peptide completely suppresses the gain in trafficking ability of GluK1(SPGluK2), indicating that the signal peptide represses receptor trafficking in a trans manner. Furthermore, we demonstrate that the signal peptide directly interacts with the amino-terminal domain (ATD) to inhibit the synaptic and surface expression of GluK1. Thus, we have uncovered a trafficking mechanism for kainate receptors and propose that the cleaved signal peptide behaves as a ligand of GluK1, through binding with the ATD, to repress forward trafficking of the receptor.

Association of TRIMCyp and TRIM5α from assam macaques leads to a functional trade-off between HIV-1 and N-MLV inhibition.

TRIM5α restricts retroviruses in a species-specific manner. Cyclophilin A was independently retrotransposed into the TRIM5 loci in different species, leading to the generation of antiviral TRIM5-cyclophilin A (TRIMCyp) proteins. Previously, we found that assam macaques express a TRIMCyp chimera (amTRIMCyp), along with a TRIM5α allelic protein (amTRIM5α). Herein, we investigated the antiviral activity of amTRIMCyp and amTRIM5α individually, as well as their interaction and joint effects. amTRIMCyp showed a divergent restriction pattern from amTRIM5α. Although both proteins potently restricted the replication of HIV-1, only amTRIM5α inhibited N-MLV. Remarkably, cellular anti-HIV-1 activity increased when amTRIMCyp and amTRIM5α were coexpressed, indicating a synergistic block of HIV-1 replication. Consistently, PMBCs from heterozygous amTRIM5α/TRIMCyp showed stronger resistance to HIV-1 infection than those from amTRIM5α/TRIM5α homozygotes. The anti-HIV-1 synergistic effect was dependent on the amTRIMCyp-amTRIM5α interaction. In contrast, amTRIMCyp completely abrogated the anti-N-MLV activity mediated by amTRIM5α, showing a dominant-negative effect, indicating that the generation of amTRIMCyp was involved in the trade-off between divergent restriction activities. Our results provide a new paradigm to study functional trade-offs mediated by allelic proteins, a theoretical basis for utilizing animal models with various TRIM5 alleles, as well as novel HIV-1 gene therapy strategies.

An all-trans-retinal-binding opsin peropsin as a potential dark-active and light-inactivated G protein-coupled receptor.

Peropsin or retinal pigment epithelium-derived rhodopsin homolog, found in many animals, belongs to the opsin family. Most opsins bind to 11-cis-retinal as a chromophore and act as light-activated G protein-coupled receptors. Some peropsins, however, bind all-trans-retinal and isomerise it into 11-cis form by light, and peropsin has been suggested to supply other visual opsins with 11-cis-retinal. Additionally, peropsin has some amino acid sequence motifs that are highly conserved among G protein-coupled opsins. Here, using chimeric mutant peropsins, we found that peropsin potentially generates an "active form" that drives G-protein signalling in the dark by binding to all-trans-retinal and that the active form photo-converts to an inactive form containing 11-cis-retinal. Comparative spectroscopic analysis demonstrated that spider peropsin exhibited catalytic efficiency for retinal photoisomerisation that was much lower than a retinal photoisomerase, squid retinochrome. The chimeric peropsins, constructed by replacing the third intracellular loop region with that of Gs- or Gi-coupled opsin, were active and drove Gs- or Gi-mediated signalling in the dark, respectively, and were inactivated upon illumination in mammalian cultured cells. These results suggest that peropsin acts as a dark-active, light-inactivated G protein-coupled receptor and is useful as a novel optogenetic tool.