Encoding Amino Acid Residues Research Articles

PMSFF: Improved Protein Binding Residues Prediction through Multi-Scale Sequence-Based Feature Fusion Strategy.

Proteins perform different biological functions through binding with various molecules which are mediated by a few key residues and accurate prediction of such protein binding residues (PBRs) is crucial for understanding cellular processes and for designing new drugs. Many computational prediction approaches have been proposed to identify PBRs with sequence-based features. However, these approaches face two main challenges: (1) these methods only concatenate residue feature vectors with a simple sliding window strategy, and (2) it is challenging to find a uniform sliding window size suitable for learning embeddings across different types of PBRs. In this study, we propose one novel framework that could apply multiple types of PBRs Prediciton task through Multi-scale Sequence-based Feature Fusion (PMSFF) strategy. Firstly, PMSFF employs a pre-trained language model named ProtT5, to encode amino acid residues in protein sequences. Then, it generates multi-scale residue embeddings by applying multi-size windows to capture effective neighboring residues and multi-size kernels to learn information across different scales. Additionally, the proposed model treats protein sequences as sentences, employing a bidirectional GRU to learn global context. We also collect benchmark datasets encompassing various PBRs types and evaluate our PMSFF approach to these datasets. Compared with state-of-the-art methods, PMSFF demonstrates superior performance on most PBRs prediction tasks.

Genome-Wide Identification and Analysis of Anthocyanidin Reductase Gene Family in Lychee (Litchi chinensis Sonn.).

Anthocyanidin reductase (ANR) is a key enzyme regulating anthocyanin synthesis and accumulation in plants. Here, lychee ANR genes were globally identified, their sequence and phylogenetic characteristics were analyzed, and their spatiotemporal expression patterns were characterized. A total of 51 ANR family members were identified in the lychee genome. The length of the encoded amino acid residues ranged from 87 aa to 289 aa, the molecular weight ranged from 9.49 KD to 32.40 KD, and the isoelectric point (pI) ranged from 4.83 to 9.33. Most of the members were acidic proteins. Most members of the LcANR family were located in the cytoplasm. The 51 LcANR family members were unevenly distributed in 11 chromosomes, and their exons and motif conserved structures were significantly different from each other. Promoters in over 90% of LcANR members contained anaerobically induced response elements, and 88% contained photoresponsive elements. Most LcANR family members had low expression in nine lychee tissues and organs (root, young leaf, bud, female flower, male flower, pericarp, pulp, seed, and calli), and some members showed tissue-specific expression patterns. The expression of one gene, LITCHI029356.m1, decreased with the increase of anthocyanin accumulation in 'Feizixiao' and 'Ziniangxi' pericarp, which was negatively correlated with pericarp coloring. The identified LcANR gene was heterologously expressed in tobacco K326, and the function of the LcANR gene was verified. This study provides a basis for the further study of LcANR function, particularly the role in lychee pericarp coloration.

Base-editing mutagenesis maps alleles to tune human T cell functions.

CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with thepotential to accelerate immunotherapy design.

Evolutionary pathways for deep-sea adaptation in marine planktonic Actinobacteriota.

The deep ocean, one of the largest ecosystems on earth, is dominated by microorganisms that are keystones in the regulation of biogeochemical cycles. However, the evolutionary pathways underlying the specific adaptations required (e.g., high pressure and low temperature) by this unique niche remain understudied. Here, we analyzed the first representatives belonging to the order Acidimicrobiales, a group of marine planktonic Actinobacteriota, that specifically inhabits the aphotic zone of the oceanic water column (>200 m). Compared with their epipelagic counterparts, deep-sea representatives showed the same evolution in genome architecture with higher GC content, longer intergenic spaces as well as higher nitrogen (N-ARSC) and lower carbon (C-ARSC) content in encoded amino acid residue side chains consistent with the higher nitrogen concentration and lower carbon concentration in deep waters compared to the photic zone. Metagenomic recruitment showed distribution patterns that allowed the description of different ecogenomic units within the three deep water-associated genera defined by our phylogenomic analyses (UBA3125, S20-B6 and UBA9410). The entire genus UBA3125 was found exclusively associated with oxygen minimum zones linked to the acquisition of genes involved in denitrification. Genomospecies of genus S20-B6 recruited in samples from both mesopelagic (200-1,000 m) and bathypelagic (1000-4,000 m) zones, including polar regions. Diversity in the genus UBA9410 was higher, with genomospecies widely distributed in temperate zones, others in polar regions, and the only genomospecies associated with abyssal zones (>4,000 m). At the functional level, groups beyond the epipelagic zone have a more complex transcriptional regulation including in their genomes a unique WhiB paralog. In addition, they showed higher metabolic potential for organic carbon and carbohydrate degradation as well as the ability to accumulate glycogen as a source of carbon and energy. This could compensate for energy metabolism in the absence of rhodopsins, which is only present in genomes associated with the photic zone. The abundance in deep samples of cytochrome P450 monooxygenases associated with the genomes of this order suggests an important role in remineralization of recalcitrant compounds throughout the water column.

Local Backbone Geometry Plays a Critical Role in Determining Conformational Preferences of Amino Acid Residues in Proteins.

The definition of the structural basis of the conformational preferences of the genetically encoded amino acid residues is an important yet unresolved issue of structural biology. In order to gain insights into this intricate topic, we here determined and compared the amino acid propensity scales for different (φ, ψ) regions of the Ramachandran plot and for different secondary structure elements. These propensities were calculated using the Chou–Fasman approach on a database of non-redundant protein chains retrieved from the Protein Data Bank. Similarities between propensity scales were evaluated by linear regression analyses. One of the most striking and unexpected findings is that distant regions of the Ramachandran plot may exhibit significantly similar propensity scales. On the other hand, contiguous regions of the Ramachandran plot may present anticorrelated propensities. In order to provide an interpretative background to these results, we evaluated the role that the local variability of protein backbone geometry plays in this context. Our analysis indicates that (dis)similarities of propensity scales between different regions of the Ramachandran plot are coupled with (dis)similarities in the local geometry. The concept that similarities of the propensity scales are dictated by the similarity of the NCαC angle and not necessarily by the similarity of the (φ, ψ) conformation may have far-reaching implications in the field.

Analysis of sequence variations in the GABA gated chloride gene associated with resistance to fipronil in Rhipicephalus (Boophilus) microplus ticks from Argentina

This study aimed to evaluate the sequence variations of the GABA-Cl gene of Rhipicephalus (Boophilus) microplus ticks from Argentina with different resistance levels to fipronil. Genomic DNA was obtained from engorged females (n = 50) of fipronil-susceptible and resistant tick field populations from five localities in northern Argentina. Tick populations were tested for fipronil resistance by in vitro larval packet test. DNA encoding amino acid residues 264–360 of the GABA-Cl was amplified by PCR. PCR products were sequenced and the translated amino acid sequences were aligned and compared among each other and with sequences of the GABA-Cl belonging to the Brazilian and Uruguayan reference strains fipronil-susceptible (Mozo) and resistant (RFSan and Juarez). All the GABA-Cl amino acid sequences obtained from the R. microplus specimens from Argentina were identical, regardless of the fipronil resistance-susceptibility status of each population. Mutations present in GABA-Cl sequence of RFSan and Juarez strains were not present in any of the populations from Argentina analyzed. These results indicate the failure of this gene as a molecular marker of resistance to fipronil, at least in the R. microplus populations from northern Argentina.

Development of a Dengue Virus Serotype-Specific Non-Structural Protein 1 Capture Immunochromatography Method.

Four serotypes of dengue virus (DENV), type 1 to 4 (DENV-1 to DENV-4), exhibit approximately 25–40% of the difference in the encoded amino acid residues of viral proteins. Reverse transcription of RNA extracted from specimens followed by PCR amplification is the current standard method of DENV serotype determination. However, since this method is time-consuming, rapid detection systems are desirable. We established several mouse monoclonal antibodies directed against DENV non-structural protein 1 and integrated them into rapid DENV detection systems. We successfully developed serotype-specific immunochromatography systems for all four DENV serotypes. Each system can detect 104 copies/mL in 15 min using laboratory and clinical isolates of DENV. No cross-reaction between DENV serotypes was observed in these DENV isolates. We also confirmed that there was no cross-reaction with chikungunya, Japanese encephalitis, Sindbis, and Zika viruses. Evaluation of these systems using serum from DENV-infected individuals indicated a serotype specificity of almost 100%. These assay systems could accelerate both DENV infection diagnosis and epidemiologic studies in DENV-endemic areas.

Artificially Edited Alleles of the Eukaryotic Translation Initiation Factor 4E1 Gene Differentially Reduce Susceptibility to Cucumber Mosaic Virus and Potato Virus Y in Tomato.

Eukaryotic translation initiation factors, including eIF4E, are susceptibility factors for viral infection in host plants. Mutation and double-stranded RNA-mediated silencing of tomato eIF4E genes can confer resistance to viruses, particularly members of the Potyvirus genus. Here, we artificially mutated the eIF4E1 gene on chromosome 3 of a commercial cultivar of tomato (Solanum lycopersicum L.) by using CRISPR/Cas9. We obtained three alleles, comprising two deletions of three and nine nucleotides (3DEL and 9DEL) and a single nucleotide insertion (1INS), near regions that encode amino acid residues important for binding to the mRNA 5' cap structure and to eIF4G. Plants homozygous for these alleles were termed 3DEL, 9DEL, and 1INS plants, respectively. In accordance with previous studies, inoculation tests with potato virus Y (PVY; type member of the genus Potyvirus) yielded a significant reduction in susceptibility to the N strain (PVYN), but not to the ordinary strain (PVYO), in 1INS plants. 9DEL among three artificial alleles had a deleterious effect on infection by cucumber mosaic virus (CMV, type member of the genus Cucumovirus). When CMV was mechanically inoculated into tomato plants and viral coat accumulation was measured in the non-inoculated upper leaves, the level of viral coat protein was significantly lower in the 9DEL plants than in the parental cultivar. Tissue blotting of microperforated inoculated leaves of the 9DEL plants revealed significantly fewer infection foci compared with those of the parental cultivar, suggesting that 9DEL negatively affects the initial steps of infection with CMV in a mechanically inoculated leaf. In laboratory tests, viral aphid transmission from an infected susceptible plant to 9DEL plants was reduced compared with the parental control. Although many pathogen resistance genes have been discovered in tomato and its wild relatives, no CMV resistance genes have been used in practice. RNA silencing of eIF4E expression has previously been reported to not affect susceptibility to CMV in tomato. Our findings suggest that artificial gene editing can introduce additional resistance to that achieved with mutagenesis breeding, and that edited eIF4E alleles confer an alternative way to manage CMV in tomato fields.

Variant analysis of SARS-CoV-2 genomes.

ObjectiveTo analyse genome variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).MethodsBetween 1 February and 1 May 2020, we downloaded 10 022 SARS CoV-2 genomes from four databases. The genomes were from infected patients in 68 countries. We identified variants by extracting pairwise alignment to the reference genome NC_045512, using the EMBOSS needle. Nucleotide variants in the coding regions were converted to corresponding encoded amino acid residues. For clade analysis, we used the open source software Bayesian evolutionary analysis by sampling trees, version 2.5.FindingsWe identified 5775 distinct genome variants, including 2969 missense mutations, 1965 synonymous mutations, 484 mutations in the non-coding regions, 142 non-coding deletions, 100 in-frame deletions, 66 non-coding insertions, 36 stop-gained variants, 11 frameshift deletions and two in-frame insertions. The most common variants were the synonymous 3037C > T (6334 samples), P4715L in the open reading frame 1ab (6319 samples) and D614G in the spike protein (6294 samples). We identified six major clades, (that is, basal, D614G, L84S, L3606F, D448del and G392D) and 14 subclades. Regarding the base changes, the C > T mutation was the most common with 1670 distinct variants.ConclusionWe found that several variants of the SARS-CoV-2 genome exist and that the D614G clade has become the most common variant since December 2019. The evolutionary analysis indicated structured transmission, with the possibility of multiple introductions into the population.

Strong Purifying Selection Is Associated with Genome Streamlining in Epipelagic Marinimicrobia.

Marine microorganisms inhabiting nutrient-depleted waters play critical roles in global biogeochemical cycles due to their abundance and broad distribution. Many of these microbes share similar genomic features including small genome size, low % G + C content, short intergenic regions, and low nitrogen content in encoded amino acid residue side chains (N-ARSC), but the evolutionary drivers of these characteristics are unclear. Here, we compared the strength of purifying selection across the Marinimicrobia, a candidate phylum which encompasses a broad range of phylogenetic groups with disparate genomic features, by estimating the ratio of nonsynonymous and synonymous substitutions (dN/dS) in conserved marker genes. Our analysis reveals that epipelagic Marinimicrobia that exhibit features consistent with genome streamlining have significantly lower dN/dS values when compared with their mesopelagic counterparts. We also found a significant positive correlation between median dN/dS values and % G + C content, N-ARSC, and intergenic region length. We did not identify a significant correlation between dN/dS ratios and estimated genome size, suggesting the strength of selection is not a primary factor shaping genome size in this group. Our findings are generally consistent with genome streamlining theory, which postulates that many genomic features of abundant epipelagic bacteria are the result of adaptation to oligotrophic nutrient conditions. Our results are also in agreement with previous findings that genome streamlining is common in epipelagic waters, suggesting that microbes inhabiting this region of the ocean have been shaped by strong selection together with prevalent nutritional constraints characteristic of this environment.

Expression and purification of the truncated duck DTMUV NS5 protein and the subcellular localization of NS5 in vitro

Duck Tembusu virus (DTMUV) non-structural protein 5 (NS5), which harbors an N-terminal methyltransferase (MTase) domain and a C-terminal RNA-dependent RNA polymerase (RdRp) domain, is central to virus replication. In this study, a sequence encoding amino acid residues 1-408 of NS5 was amplified and ligated into a pET32a vector to generate a pET32a-NS51-408 expression vector. Recombinant protein was expressed, purified, and used to arise a mouse anti-NS5 specific polyclonal antibody (DTMUV-NS51-408 mPAb). Indirect immunofluorescence assays indicated that NS5 protein localized within the cytoplasmic region of DTMUV-infected or transfected cells; the localization was not affected in the presence of a nuclear export inhibitor. This study provides the first demonstration that, in contrast to the data reported for NS5 proteins of Dengue and Yellow fever virus, DTMUV NS5 had slight nuclear localization activity.

Identification of a specific domain of Porphyromonas gingivalis Hgp44 responsible for adhesion to Treponema denticola.

Interaction between two periodontal pathogens, Porphyromonas gingivalis and Treponema denticola, contributes to plaque biofilm formation. Porphyromonas gingivalis forms aggregates with T. denticola through its adhesion/hemagglutinin domain (Hgp44). In this study, we investigated the specific domain of P. gingivalis Hgp44 responsible for adhesion to T. denticola using expression vectors harboring P. gingivalis Hgp44 DNA sequences encoding amino acid residues 1-419. Six plasmids harboring fragments in this region were generated by PCR amplification and self-ligation, and recombinant proteins r-Hgp44 (residues 1-419), r-Hgp441 (residues 1-124), r-Hgp442 (1-199), r-Hgp443 (1-316), r-Hgp444 (199-419), r-Hgp445 (124-198) and r-Hgp446 (199-316) were produced, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. r-Hgp44, r-Hgp443 and r-Hgp446 showed greater adhesion to T. denticola sonicates than the control, as determined by enzyme-linked immunosorbent assay. r-Hgp446 reduced the coaggregation of P. gingivalis and T. denticola. Scanning electron and confocal laser scanning microscopy analyses revealed that r-Hgp446 reduced dual-species biofilm formation. Our results indicate that residues 199-316 of P. gingivalis Hgp44 are mainly responsible for adhesion to T. denticola; inhibiting this domain could potentially disrupt periodontopathic biofilm formation and maturation.

Insights into the phylogenetic and molecular evolutionary histories of Fad and Elovl gene families in Actiniaria.

The biosynthesis of long‐chain polyunsaturated fatty acids (LC‐PUFAs, ≥ C20) is reliant on the action of desaturase and elongase enzymes, which are encoded by the fatty acyl desaturase (Fad) and elongation of very long‐chain fatty acid (Elovl) gene families, respectively. In Metazoa, research investigating the distribution and evolution of these gene families has been restricted largely to Bilateria. Here, we provide insights into the phylogenetic and molecular evolutionary histories of the Fad and Elovl gene families in Cnidaria, the sister phylum to Bilateria. Four model cnidarian genomes and six actiniarian transcriptomes were interrogated. Analysis of the fatty acid composition of a candidate cnidarian species, Actinia tenebrosa, was performed to determine the baseline profile of this species. Phylogenetic analysis revealed lineage‐specific gene duplication in actiniarians for both the Fad and Elovl gene families. Two distinct cnidarian Fad clades clustered with functionally characterized Δ5 and Δ6 proteins from fungal and plant species, respectively. Alternatively, only a single cnidarian Elovl clade clustered with functionally characterized Elovl proteins (Elovl4), while two additional clades were identified, one actiniarian‐specific (Novel ElovlA) and the another cnidarian‐specific (Novel ElovlB). In actiniarians, selection analyses revealed pervasive purifying selection acting on both gene families. However, codons in the Elovl gene family show patterns of nucleotide variation consistent with the action of episodic diversifying selection following gene duplication events. Significantly, these codons may encode amino acid residues that are functionally important for Elovl proteins to target and elongate different precursor fatty acids. In A. tenebrosa, the fatty acid analysis revealed an absence of LC‐PUFAs > C20 molecules and implies that the Elovl enzymes are not actively contributing to the elongation of these LC‐PUFAs. Overall, this study has revealed that actiniarians possess Fad and Elovl genes required for the biosynthesis of some LC‐PUFAs, and that these genes appear to be distinct from bilaterians.

Genome-wide survey of codons under diversifying selection in a highly recombining bacterial species, Helicobacter pylori.

Selection has been a central issue in biology in eukaryotes as well as prokaryotes. Inference of selection in recombining bacterial species, compared with clonal ones, has been a challenge. It is not known how codons under diversifying selection are distributed along the chromosome or among functional categories or how frequently such codons are subject to mutual homologous recombination. Here, we explored these questions by analysing genes present in >90% among 29 genomes of Helicobacter pylori, one of the bacterial species with the highest mutation and recombination rates. By a method for recombining sequences, we identified codons under diversifying selection (dN/dS > 1), which were widely distributed and accounted for ∼0.2% of all the codons of the genome. The codons were enriched in genes of host interaction/cell surface and genome maintenance (DNA replication, recombination, repair, and restriction modification system). The encoded amino acid residues were sometimes found adjacent to critical catalytic/binding residues in protein structures. Furthermore, by estimating the intensity of homologous recombination at a single nucleotide level, we found that these codons appear to be more frequently subject to recombination. We expect that the present study provides a new approach to population genomics of selection in recombining prokaryotes.

English

microRNAs are small RNA molecules that inhibit the translation of target genes. microRNA binding sites are located in the untranslated regions as well as in the coding domains. We describe TmiRUSite and TmiROSite scripts developed using python as tools for the extraction of nucleotide sequences for miRNA binding sites with their encoded amino acid residue sequences. The scripts allow for retrieving a set of additional sequences at left and at right from the binding site. The scripts presents all received data in table formats that are easy to analyse further. The predicted data finds utility in molecular and evolutionary biology studies. They find use in studying miRNA binding sites in animals and plants.AvailabilityTmiRUSite and TmiROSite scripts are available for free from authors upon request and at https: //sites.google.com/site/malaheenee/downloads for download

Amino acid diversity of antigenic sites of Iranian type O foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is a major cause of endemic outbreaks in livestock in Iran. In this study, clinical field samples of foot-and-mouth disease virus were collected from an outbreak in Khorasan Razavi Province during April and August of 2010, and subjected to indirect sandwich ELISA and RT-PCR. The viral serotype circulating during the period was confirmed to be type O. The virus was then genetically characterized for its complete P1 genomic sequences to compare with nine corresponding nucleotide sequences of representative foot-and-mouth disease viruses (FMDVs) registered in the GenBank. The P1-coding region was 2208 nucleotides in length with 736 encoded amino acid residues. Phylogenetic analysis revealed two major lineages of A (with three additional clusters) and B. Iranian field isolate was grouped within cluster I, most closely related to Pakistani strains PAK/39/2008 and PAK/29/2008 sharing 98.37 and 98.1% amino acid identity, respectively, demonstrating the close epidemiological links between countries in the region. In contrast, our isolate showed low amino acid identity with Italian isolate O-2-Brescia (93.48%) and Argentinean isolate O1 Caseros (93.75%). Based on multiple sequence alignments, comparison of sequences showed that the characteristic amino acid mutations were found in the VP1, VP2 and VP3 proteins of isolated virus. This article is the first to report on the complete P1 genomic characterization of type O FMDV circulating in Iran

Apple miRNAs and tasiRNAs with novel regulatory networks

BackgroundMicroRNAs (miRNAs) and their regulatory functions have been extensively characterized in model species but whether apple has evolved similar or unique regulatory features remains unknown.ResultsWe performed deep small RNA-seq and identified 23 conserved, 10 less-conserved and 42 apple-specific miRNAs or families with distinct expression patterns. The identified miRNAs target 118 genes representing a wide range of enzymatic and regulatory activities. Apple also conserves two TAS gene families with similar but unique trans-acting small interfering RNA (tasiRNA) biogenesis profiles and target specificities. Importantly, we found that miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. These miRNA target sites are differentially conserved among MYBs, which is largely influenced by the location and conservation of the encoded amino acid residues in MYB factors. Finally, we found that 10 of the 19 miR828-targeted MYBs undergo small interfering RNA (siRNA) biogenesis at the 3' cleaved, highly divergent transcript regions, generating over 100 sequence-distinct siRNAs that potentially target over 70 diverse genes as confirmed by degradome analysis.ConclusionsOur work identified and characterized apple miRNAs, their expression patterns, targets and regulatory functions. We also discovered that three miRNAs and the ensuing siRNAs exploit both conserved and divergent sequence features of MYB genes to initiate distinct regulatory networks targeting a multitude of genes inside and outside the MYB family.

Consensus Substrate Sequence for Protein-tyrosine Phosphatase Receptor Type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) has multiple substrate proteins, including G protein-coupled receptor kinase-interactor 1 (Git1), membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190RhoGAP). We have identified a dephosphorylation site at Tyr-1105 of p190RhoGAP; however, the structural determinants employed for substrate recognition of Ptprz have not been fully defined. In the present study, we revealed that Ptprz selectively dephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 and Tyr-858 by in vitro and cell-based assays. Of note, the dephosphorylation of the Magi1 Tyr-858 site required PDZ domain-mediated interaction between Magi1 and Ptprz in the cellular context. Alignment of the primary sequences surrounding the target phosphotyrosine residue in these three substrates showed considerable similarity, suggesting a consensus motif for recognition by Ptprz. We then estimated the contribution of surrounding individual amino acid side chains to the catalytic efficiency by using fluorescent peptides based on the Git1 Tyr-554 sequence in vitro. The typical substrate motif for the catalytic domain of Ptprz was deduced to be Glu/Asp-Glu/Asp-Glu/Asp-Xaa-Ile/Val-Tyr(P)-Xaa (Xaa is not an acidic residue). Intriguingly, a G854D substitution of the Magi1 Tyr-858 site matching better to the motif sequence turned this site to be susceptible to dephosphorylation by Ptprz independent of the PDZ domain-mediated interaction in cells. Furthermore, we found by database screening that the substrate motif is present in several proteins, including paxillin at Tyr-118, its major phosphorylation site. Expectedly, we verified that Ptprz efficiently dephosphorylates paxillin at this site in cells. Our study thus provides key insights into the molecular basis for the substrate recognition of Ptprz.

RB1CC1 Protein Positively Regulates Transforming Growth Factor-β Signaling through the Modulation of Arkadia E3 Ubiquitin Ligase Activity

Transforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators, of which Smad7, c-Ski, and SnoN play a pivotal role in its negative regulation. Arkadia is a RING-type E3 ubiquitin ligase that targets these negative regulators for degradation to enhance TGF-β signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-β signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-β signaling, and knockdown of endogenous RB1CC1 attenuated TGF-β-induced expression of target genes as well as TGF-β-induced cytostasis. RB1CC1 down-regulated the protein levels of c-Ski but not SnoN by enhancing the activity of Arkadia E3 ligase toward c-Ski. Substrate selectivity is primarily attributable to the physical interaction of RB1CC1 with substrates, suggesting its role as a scaffold protein. RB1CC1 thus appears to play a unique role as a modulator of TGF-β signaling by restricting substrate specificity of Arkadia.

Regulatory Roles of Heterogeneous Nuclear Ribonucleoprotein M and Nova-1 Protein in Alternative Splicing of Dopamine D2 Receptor Pre-mRNA

The dopamine D2 receptor (D2R) plays a crucial role in the regulation of diverse key physiological functions, including motor control, reward, learning, and memory. This receptor is present in vivo in two isoforms, D2L and D2S, generated from the same gene by alternative pre-mRNA splicing. Each isoform has a specific role in vivo, underlining the importance of a strict control of its synthesis, yet the molecular mechanism modulating alternative D2R pre-mRNA splicing has not been completely elucidated. Here, we identify heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a key molecule controlling D2R splicing. We show that binding of hnRNP M to exon 6 inhibited the inclusion of this exon in the mRNA. Importantly, the splicing factor Nova-1 counteracted hnRNP M effects on D2R pre-mRNA splicing. Indeed, mutations of the putative Nova-1-binding site on exon 6 disrupted Nova-1 RNA assembly and diminished the inhibitory effect of Nova-1 on hnRNP M-dependent exon 6 exclusion. These results identify Nova-1 and hnRNP M as D2R pre-mRNA-binding proteins and show their antagonistic role in the alternative splicing of D2R pre-mRNA.