Phosphoserine aminotransferase deficiency diagnosed by whole-exome sequencing and LC-MS/MS reanalysis: A case report and review of literature.

BACKGROUND: Phosphoserine aminotransferase deficiency (PSATD) is an autosomal recessive disorder associated with hypertonia, psychomotor retardation, and acquired microcephaly. Patients with PSATD have low concentrations of serine in plasma and cerebrospinal fluid. METHODS: We reported a 2-year-old female child with developmental delay, dyskinesia, and microcephaly. LC-MS/MS was used to detect amino acid concentration in the blood and whole-exome sequencing (WES) was used to identify the variants. PolyPhen-2 web server and PyMol were used to predict the pathogenicity and changes in the 3D model molecular structure of protein caused by variants. RESULTS: WES demonstrated compound heterozygous variants in PSAT1, which is associated with PSATD, with a paternal likely pathogenic variant (c.235G>A, Gly79Arg) and a maternal likely pathogenic variant (c.43G>C, Ala15Pro). Reduced serine concentration in LC-MS/MS further confirmed the diagnosis of PSATD in this patient. CONCLUSIONS: Our findings demonstrate the importance of WES combined with LC-MS/MS reanalysis in the diagnosis of genetic diseases and expand the PSAT1 variant spectrum in PSATD. Moreover, we summarize all the cases caused by PSAT1 variants in the literature. This case provides a vital reference for the diagnosis of future cases.

Association of dopamine receptor D3 polymorphism with Levodopa-induced Dyskinesia: A study on Parkinson's disease patients from India.

INTRODUCTION: Levodopa-induced dyskinesia (LID) is a debilitating motor feature in a subset of patients with Parkinson's disease (PD) after prolonged therapeutic administration of levodopa. Preliminary animal and human studies are suggestive of a key role of dopamine type 3 (D3) receptor polymorphism (Ser9Gly; rs6280) in LID. Its contribution to development of LID among Indian PD patients has remained relatively unexplored and merits further investigation. METHODS AND MATERIALS: 200 well-characterised PD patients (100 without LID and 100 with LID) and 100 age-matched healthy controls were recruited from the outpatient department of Institute of Neurosciences Kolkata. MDS-UPDRS (Unified Parkinson's Disease Rating Scale from International Movement Disorder Society) Part III and AIMS (abnormal involuntary movement scale) were performed for estimation of severity of motor features and LID respectively in the ON state of the disease. Participants were analysed for the presence of Ser9Gly single nucleotide variant (SNV) (rs6280) by polymerase chain reaction followed by restriction fragment length polymorphism techniques. RESULTS: The frequency of AA genotype (serine type) was more frequently present in PD patients with LID compared to PD patients without LID (50 % vs 28 %; P = 0.002; OR = 2.57, 95 % CI: 1.43 - 4.62). The abnormal involuntary movement scale score was significantly higher in PD patients with AA genotype compared to carriers of glycine allele (AG + GG) (4.08 ± 3.35; P = 0.002). CONCLUSION: We observed a significant association of serine type SNV (rs6280) in D3 receptor gene in a cohort of PD patients with LID from India. More severe motor severity was found in patients with glycine substitution of the same SNV. The current study emphasised the role of D3 receptor in the pathogenesis of LID.

Transcriptome Study of Bursaphelenchus xylophilus Treated with Fomepizole Reveals a Serine/Threonine-Protein Phosphatase Gene that Is Substantially Linked with Vitality and Pathogenicity.

Bursaphelenchus xylophilus, the pine wood nematode (PWN), is the causal agent of pine wilt disease (PWD), which causes enormous economic loss annually. According to our previous research, fomepizole, as a selective inhibitor of PWN alcohol dehydrogenase (ADH), has the potential to be a preferable lead compound for developing novel nematicides. However, the underlying molecular mechanism is still unclear. The result of molecular docking showed that the stronger interactions between fomepizole and PWN ADH at the active site of ADH were attributed to hydrogen bonds. Low-dose fomepizole had a substantial negative impact on the egg hatchability, development, oviposition, and lifespan of PWN. Transcriptome analysis indicated that 2,124 upregulated genes and 490 downregulated genes in fomepizole-treated PWN were obtained. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes indicated that fomepizole could be involved in controlling PWN vitality mainly by regulating key signaling pathways, such as the ribosome, hippo signaling pathway, and lysosome. Remarkably, the results of RNA interference indicated that the downregulated serine/threonine-protein phosphatase gene (stpp) could reduce the egg hatchability, development, oviposition, and lifespan of PWN, which was closely similar to the consequences of nematodes with low-dose fomepizole treatment. In addition, the silencing of stpp resulted in weakness of PWN pathogenicity, which indicated that stpp could be a potential drug target to control PWN.

Phylogeny and Expansion of Serine/Threonine Kinases in Phagocytotic Bacteria in the Phylum Planctomycetota.

The recently isolated bacterium "Candidatus Uabimicrobium amorphum" is the only known prokaryote that can engulf other bacterial cells. Its proteome contains a high fraction of proteins involved in signal transduction systems, which is a feature normally associated with multicellularity in eukaryotes. Here, we present a protein-based phylogeny which shows that "Ca. Uabimicrobium amorphum" represents an early diverging lineage that clusters with the Saltatorellus clade within the phylum Planctomycetota. A gene flux analysis indicated a gain of 126 protein families for signal transduction functions in "Ca. Uabimicrobium amorphum", of which 66 families contained eukaryotic-like Serine/Threonine kinases with Pkinase domains. In total, we predicted 525 functional Serine/Threonine kinases in "Ca. Uabimicrobium amorphum", which represent 8% of the proteome and is the highest fraction of Serine/Threonine kinases in a bacterial proteome. The majority of Serine/Threonine kinases in this species are membrane proteins and 30% contain long, tandem arrays of WD40 or TPR domains. The pKinase domain was predicted to be located in the cytoplasm, while the WD40 and TPR domains were predicted to be located in the periplasm. Such domain combinations were also identified in the Serine/Threonine kinases of other species in the Planctomycetota, although in much lower abundances. A phylogenetic analysis of the Serine/Threonine kinases in the Planctomycetota inferred from the Pkinase domain alone provided support for lineage-specific expansions of the Serine/Threonine kinases in "Ca. Uabimicrobium amorphum". The results imply that expansions of eukaryotic-like signal transduction systems are not restricted to multicellular organisms, but have occurred in parallel in prokaryotes with predatory lifestyles and phagocytotic-like behaviors.

Elucidation of productive alanine recognition mechanism by Escherichia coli alanyl-tRNA synthetase.

Alanyl-tRNA synthetase (AlaRS) incorrectly recognizes both a slightly smaller glycine and a slightly larger serine in addition to alanine, and the probability of incorrect identification is extremely low at 1/300 and 1/170, respectively. Alanine is the second smallest amino acid after glycine; however, the mechanism by which AlaRS specifically identifies small differences in side chains with high accuracy remains unknown. In this study, using a malachite green assay, we aimed to elucidate the alanine recognition mechanism of a fragment (AlaRS368N) containing only the amino acid activation domain of Escherichia coli AlaRS. This method quantifies monophosphate by decomposing pyrophosphate generated during aminoacyl-AMP production. AlaRS368N produced far more pyrophosphate when glycine or serine was used as a substrate than when alanine was used. Among several mutants tested, an AlaRS mutant in which the widely conserved aspartic acid at the 235th position (D235) near the active center was replaced with glutamic acid (D235E) increased pyrophosphate release for the alanine substrate, compared to that from glycine and serine. These results suggested that D235 is optimal for AlaRS to specifically recognize alanine. Alanylation activities of an RNA minihelix by the mutants of valine at the 214th position (V214) of another fragment (AlaRS442N), which is the smallest AlaRS with alanine charging activity, suggest the existence of the van der Waals-like interaction between the side chain of V214 and the methyl group of the alanine substrate.

The association of TMPRSS6 gene polymorphism with iron status in Egyptian children (a pilot study).

Several studies have shown association of single nucleotide polymorphisms (SNPs) of hepcidin regulatory pathways genes with impaired iron status. The most common is in the TMPRSS6 gene. In Africa, very few studies have been reported. We aimed to investigate the correlation between the common SNPs in the transmembrane protease, serine 6 (TMPRSS6) gene and iron indicators in a sample of Egyptian children for identifying the suitable candidate for iron supplementation.Patients and methods One hundred and sixty children aged 5-13 years were included & classified into iron deficient, iron deficient anemia and normal healthy controls. All were subjected to assessment of serum iron, serum ferritin, total iron binding capacity, complete blood count, reticulocyte count, serum soluble transferrin receptor and serum hepcidin. Molecular study of TMPRSS6 genotyping polymorphisms (rs4820268, rs855791 and rs11704654) were also evaluated.Results There was an association of iron deficiency with AG of rs855791 SNP, (P = 0.01). The minor allele frequency for included children were 0.43, 0.45 & 0.17 for rs4820268, rs855791 & rs11704654 respectively. Genotype GG of rs4820268 expressed the highest hepcidin gene expression fold, the lowest serum ferroportin & iron store compared to AA and AG genotypes (p = 0.05, p = 0.05, p = 0.03 respectively). GG of rs855791 had lower serum ferritin than AA (p = 0.04), lowest iron store & highest serum hepcidin compared to AA and AG genotypes (p = 0.04, p = 0.01 respectively). Children having CC of rs11704654 had lower level of hemoglobin, serum ferritin and serum hepcidin compared with CT genotype (p = 0.01, p = 0.01, p = 0.02) respectively.Conclusion Possible contribution of SNPs (rs855791, rs4820268 and rs11704654) to low iron status.

Mitogenomic Characterization and Phylogenetic Placement of African Hind, Cephalopholis taeniops: Shedding Light on the Evolution of Groupers (Serranidae: Epinephelinae).

The global exploration of evolutionary trends in groupers, based on mitogenomes, is currently underway. This research extensively investigates the structure of and variations in Cephalopholis species mitogenomes, along with their phylogenetic relationships, focusing specifically on Cephalopholis taeniops from the Eastern Atlantic Ocean. The generated mitogenome spans 16,572 base pairs and exhibits a gene order analogous to that of the ancestral teleost's, featuring 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and an AT-rich control region. The mitogenome of C. taeniops displays an AT bias (54.99%), aligning with related species. The majority of PCGs in the mitogenome initiate with the start codon ATG, with the exceptions being COI (GTG) and atp6 (TTG). The relative synonymous codon usage analysis revealed the maximum abundance of leucine, proline, serine, and threonine. The nonsynonymous/synonymous ratios were <1, which indicates a strong negative selection among all PCGs of the Cephalopholis species. In C. taeniops, the prevalent transfer RNAs display conventional cloverleaf secondary structures, except for tRNA-serine (GCT), which lacks a dihydrouracil (DHU) stem. A comparative examination of conserved domains and sequence blocks across various Cephalopholis species indicates noteworthy variations in length and nucleotide diversity. Maximum likelihood, neighbor-joining, and Bayesian phylogenetic analyses, employing the concatenated PCGs and a combination of PCGs + rRNAs, distinctly separate all Cephalopholis species, including C. taeniops. Overall, these findings deepen our understanding of evolutionary relationships among serranid groupers, emphasizing the significance of structural considerations in mitogenomic analyses.

The serine synthesis pathway drives osteoclast differentiation through epigenetic regulation of NFATc1 expression.

Bone-resorbing osteoclasts are vital for postnatal bone health, as increased differentiation or activity results in skeletal pathologies such as osteoporosis. The metabolism of mature osteoclasts differs from their progenitor cells, but whether the observed metabolic changes are secondary to the altered cell state or actively drive the process of cell differentiation is unknown. Here, we show that transient activation of the serine synthesis pathway (SSP) is essential for osteoclastogenesis, as deletion of the rate-limiting enzyme phosphoglycerate dehydrogenase in osteoclast progenitors impairs their differentiation and results in increased bone mass. In addition, pharmacological phosphoglycerate dehydrogenase inhibition abrogated bone loss in a mouse model of postmenopausal osteoporosis by blocking bone resorption. Mechanistically, SSP-derived α-ketoglutarate is necessary for histone demethylases that remove repressive histone methylation marks at the nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1) gene locus, thereby inducing NFATc1 expression and consequent osteoclast maturation. Taken together, this study reveals a metabolic-epigenetic coupling mechanism that directs osteoclast differentiation and suggests that the SSP can be therapeutically targeted to prevent osteoporotic bone loss.

Defining the impact of flavivirus envelope protein glycosylation site mutations on sensitivity to broadly neutralizing antibodies.

Antibodies targeting an envelope dimer epitope (EDE) cross-neutralize Zika virus (ZIKV) and dengue virus (DENV) and have thus inspired an epitope-focused vaccine design. There are two EDE antibody subclasses (EDE1, EDE2) distinguished by their dependence on viral envelope protein N-linked glycosylation at position N153 (DENV) or N154 (ZIKV) for binding. Here, we determined how envelope glycosylation site mutations affect neutralization by EDE and other broadly neutralizing antibodies. Consistent with structural studies, mutations abolishing the N153/N154 glycosylation site increased DENV and ZIKV sensitivity to neutralization by EDE1 antibodies. Surprisingly, despite their location at predicted contact sites, these mutations also increased sensitivity to EDE2 antibodies. Moreover, despite preserving the glycosylation site motif (N-X-S/T), substituting the threonine at ZIKV envelope residue 156 with a serine resulted in loss of glycan occupancy accompanied with increased neutralization sensitivity to EDE antibodies. For DENV, the presence of a serine instead of a threonine at envelope residue 155 retained glycan occupancy, but nonetheless increased sensitivity to EDE antibodies, in some cases to a similar extent as mutation at N153, which abolishes glycosylation. Envelope glycosylation site mutations also increased ZIKV and DENV sensitivity to other non-EDE broadly neutralizing antibodies, but had limited effects on ZIKV- or DENV-specific antibodies. Thus, envelope protein glycosylation is context-dependent and modulates the potency of broadly neutralizing antibodies in a manner not predicted by existing structures. Manipulating envelope protein glycosylation could be a novel strategy for engineering vaccine antigens to elicit antibodies that broadly neutralize ZIKV and DENV.IMPORTANCEAntibodies that potently cross-neutralize Zika (ZIKV) and dengue (DENV) viruses are attractive to induce via vaccination to protect against these co-circulating flaviviruses. Structural studies have shown that viral envelope protein glycosylation is important for binding by one class of these so-called broadly neutralizing antibodies, but less is known about its effect on neutralization. Here, we investigated how envelope protein glycosylation site mutations impact the potency of broadly neutralizing antibodies against ZIKV and DENV. We found that glycan occupancy was not always predicted by an intact N-X-S/T sequence motif. Moreover, envelope protein glycosylation site mutations alter the potency of broadly neutralizing antibodies in a manner unexpected from their predicted binding mechanism as determined by existing structures. We therefore highlight the complex role and determinants of envelope protein glycosylation that should be considered in the design of vaccine antigens to elicit broadly neutralizing antibodies.

Engineering serine hydroxymethyltransferases for efficient synthesis of L-serine in Escherichia coli.

L-serine is a high-value amino acid widely used in the food, medicine, and cosmetic industries. However, the low yield of L-serine has limited its industrial production. In this study, a cellular factory for efficient synthesis of L-serine was obtained by engineering the serine hydroxymethyltransferases (SHMT). Firstly, after screening the SHMT from Alcanivorax dieselolei by genome mining, a mutant AdSHMTE266M with high thermal stability was identified through rational design. Subsequently, an iterative saturating mutant library was constructed by using coevolutionary analysis, and a mutant AdSHMTE160L/E193Q with enzyme activity 1.35 times higher than AdSHMT was identified. Additionally, the target protein AdSHMTE160L/E193Q/E266M was efficiently overexpressed by improving its mRNA stability. Finally, combining the substrate addition strategy and system optimization, the optimized strain BL21/pET28a-AdSHMTE160L/E193Q/E266M-5'UTR-REP3S16 produced 106.06 g/L L-serine, which is the highest production to date. This study provides new ideas and insights for the engineering design of SHMT and the industrial production of L-serine.

Structural and mechanistic insights into ALS patient derived mutations in D-amino acid oxidase.

The D-amino acid oxidase protein modulates neurotransmission by controlling the levels of D-serine, a co-agonist of N-methyl-D-aspartate receptors. Mutations in the DAO gene have been associated with ALS, with some studies reporting pathogenic mechanisms of the R199W mutation. We have characterized two novel mutations R38H and Q201R found in ALS patients and report certain novel findings related to the R199W mutation. We report the first instance of crystal structure analysis of a patient-derived mutant of DAO, R38H, solved at 2.10 Å. The structure revealed significant perturbations and altered binding with the cofactor (FAD) and the inhibitor benzoate, supported by biochemical assays. Q201R-DAO also exhibited significantly lower ligand binding efficiency. Furthermore, kinetic analysis across all variants revealed reduced oxidase activity and substrate binding. Notably, R38H-DAO exhibited near-WT activity only at high substrate concentrations, while R199W-DAO and Q201R-DAO displayed drastic activity reduction. Additionally, structural perturbations were inferred for R199W-DAO and Q201R-DAO, evident by the higher oligomeric state in the holoenzyme form. We also observed thermal instability in case of R199W-DAO mutant. We hypothesize that the mutant enzymes may be rendered non-functional in a cellular context, potentially leading to NMDAR-associated excitotoxicity. The study provides novel insights into structural and functional aspects of DAO mutations in ALS.

Biochemical and cellular studies of three human 3-phosphoglycerate dehydrogenase variants responsible for pathological reduced L-serine levels.

In the brain, the non-essential amino acid L-serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3-phosphoglycerate: among the different roles played by this amino acid, it can be converted into D-serine and glycine, the two main co-agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate-limiting step of this pathway), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L-serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single-point variants responsible for hPHGDH deficiency and Neu-Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3-phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L-serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L-serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches.

Integrated Metabolomics, Transcriptome and Functional Analysis Reveal Key Genes Are Involved in Tree Age-Induced Amino Acid Accumulation in Torreya grandis Nuts.

Torreya grandis is native Chinese tree species of economic significance, renowned for its long lifespan and the rich nutritional value of its nuts. In this study, we analyzed the morphological characteristics, metabolites, associated gene expressions, and regulatory mechanism in nuts from young (10 years old) and old (1000 years old) T. grandis trees. We observed that the length, width, and weight of nuts from older trees were considerably greater than those from younger trees. Metabolomic analysis revealed that the concentrations of 18 amino acids and derivatives (including histidine and serine) in nuts from older trees were markedly higher than those in nuts from younger trees. Transcriptome and metabolomic correlation analysis identified 16 genes, including TgPK (pyruvate kinase), TgGAPDH (glyceraldehyde 3-phosphate dehydrogenase), and others, which exhibit higher expression levels in older trees compared to younger trees, as confirmed by qRT-PCR. These genes are associated with the biosynthesis of histidine, glutamic acid, tryptophan, and serine. Transient expression of TgPK in tobacco led to increased pyruvate kinase activity and amino acid content (histidine, tryptophan, and serine). Additionally, dual-luciferase assays and yeast one-hybrid results demonstrated that TgWRKY21 positively regulates TgPK expression by directly binding to the TgPK promoter. These findings not only demonstrate the nutritional differences between nuts from young and old trees but also offer fresh insights into the development of nutritional sources and functional components based on nuts from old trees, enriching our understanding of the potential benefits of utilizing nuts from older trees.

STAT3 phosphorylation at serine 727 activates specific genetic programs and promotes clear cell renal cell carcinoma (ccRCC) aggressiveness.

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor mainly activated by phosphorylation in either tyrosine 705 (Y705) or serine 727 (S727) residues that regulates essential processes such as cell differentiation, apoptosis inhibition, or cell survival. Aberrant activation of STAT3 has been related to development of nearly 50% of human cancers including clear cell renal cell carcinoma (ccRCC). In fact, phosho-S727 (pS727) levels correlate with overall survival of ccRCC patients. With the aim to elucidate the contribution of STAT3 phosphorylation in ccRCC development and progression, we have generated human-derived ccRCC cell lines carrying STAT3 Y705 and S727 phosphomutants. Our data show that the phosphomimetic substitution Ser727Asp facilitates a pro-tumoral phenotype in vitro, in a Y705-phosphorylation-independent manner. Moreover, we describe that STAT3 phosphorylation state determines the expression of different subsets of target genes associated with distinct biological processes, being pS727-dependent genes the most related to cellular hallmarks of cancer. In summary, the present study constitutes the first analysis on the role of overall STAT3 phosphorylation state in ccRCC and demonstrates that pS727 promotes the expression of a specific subset of target genes that might be clinically relevant as novel biomarkers and potential therapeutic targets for ccRCC.

PRO-IP-seq tracks molecular modifications of engaged Pol II complexes at nucleotide resolution.

RNA Polymerase II (Pol II) is a multi-subunit complex that undergoes covalent modifications as transcription proceeds through genes and enhancers. Rate-limiting steps of transcription control Pol II recruitment, site and degree of initiation, pausing duration, productive elongation, nascent transcript processing, transcription termination, and Pol II recycling. Here, we develop Precision Run-On coupled to Immuno-Precipitation sequencing (PRO-IP-seq), which double-selects nascent RNAs and transcription complexes, and track phosphorylation of Pol II C-terminal domain (CTD) at nucleotide-resolution. We uncover precise positional control of Pol II CTD phosphorylation as transcription proceeds from the initiating nucleotide (+1 nt), through early (+18 to +30 nt) and late (+31 to +60 nt) promoter-proximal pause, and into productive elongation. Pol II CTD is predominantly unphosphorylated from initiation until the early pause-region, whereas serine-2- and serine-5-phosphorylations are preferentially deposited in the later pause-region. Upon pause-release, serine-7-phosphorylation rapidly increases and dominates over the region where Pol II assembles elongation factors and accelerates to its full elongational speed. Interestingly, tracking CTD modifications upon heat-induced transcriptional reprogramming demonstrates that Pol II with phosphorylated CTD remains paused on thousands of heat-repressed genes. These results uncover dynamic Pol II regulation at rate-limiting steps of transcription and provide a nucleotide-resolution technique for tracking composition of engaged transcription complexes.

Multiplexed assay of variant effect reveals residues of functional importance in the BRCA1 coiled-coil and serine cluster domains.

Delineating functionally normal variants from functionally abnormal variants in tumor suppressor proteins is critical for cancer surveillance, prognosis, and treatment options. BRCA1 is a protein that has many variants of uncertain significance which are not yet classified as functionally normal or abnormal. In vitro functional assays can be used to identify the functional impact of a variant when the variant has not yet been categorized through clinical observation. Here we employ a homology-directed repair (HDR) reporter assay to evaluate over 300 missense and nonsense BRCA1 variants between amino acid residues 1280 and 1576, which encompasses the coiled-coil and serine cluster domains. Functionally abnormal variants tended to cluster in residues known to interact with PALB2, which is critical for homology-directed repair. Multiplexed results were confirmed by singleton assay and by ClinVar database variant interpretations. Comparison of multiplexed results to designated benign or likely benign or pathogenic or likely pathogenic variants in the ClinVar database yielded 100% specificity and 100% sensitivity of the multiplexed assay. Clinicians can reference the results of this functional assay for help in guiding cancer treatment and surveillance options. These results are the first to evaluate this domain of BRCA1 using a multiplexed approach and indicate the importance of this domain in the DNA repair process.

Targeting alternative splicing as a new cancer immunotherapy-phosphorylation of serine arginine-rich splicing factor (SRSF1) by SR protein kinase 1 (SRPK1) regulates alternative splicing of PD1 to generate a soluble antagonistic isoform that prevents T cell exhaustion.

BACKGROUND: Regulation of alternative splicing is a new therapeutic approach in cancer. The programmed cell death receptor 1 (PD-1) is an immunoinhibitory receptor expressed on immune cells that binds to its ligands, PD-L1 and PD-L2 expressed by cancer cells forming a dominant immune checkpoint pathway in the tumour microenvironment. Targeting this pathway using blocking antibodies (nivolumab and pembrolizumab) is the mainstay of anti-cancer immunotherapies, restoring the function of exhausted T cells. PD-1 is alternatively spliced to form isoforms that are either transmembrane signalling receptors (flPD1) that mediate T cell death by binding to the ligand, PD-L1 or an alternatively spliced, soluble, variant that lacks the transmembrane domain. METHODS: We used PCR and western blotting on primary peripheral blood mononuclear cells (PBMCs) and Jurkat T cells, IL-2 ELISA, flow cytometry, co-culture of melanoma and cholangiocarcinoma cells, and bioinformatics analysis and molecular cloning to examine the mechanism of splicing of PD1 and its consequence. RESULTS: The soluble form of PD-1, generated by skipping exon 3 (∆Ex3PD1), was endogenously expressed in PBMCs and T cells and prevents cancer cell-mediated T cell repression. Multiple binding sites of SRSF1 are adjacent to PD-1 exon 3 splicing sites. Overexpression of phosphomimic SRSF1 resulted in preferential expression of flPD1. Inhibition of SRSF1 phosphorylation both by SRPK1 shRNA knockdown and by a selective inhibitor, SPHINX31, resulted in a switch in splicing to ∆Ex3PD1. Cholangiocarcinoma cell-mediated repression of T cell IL-2 expression was reversed by SPHINX31 (equivalent to pembrolizumab). CONCLUSIONS: These results indicate that switching of the splicing decision from flPD1 to ∆Ex3PD1 by targeting SRPK1 could represent a potential novel mechanism of immune checkpoint inhibition in cancer.

Identification of the lipodepsipeptide selethramide encoded in a giant nonribosomal peptide synthetase from a Burkholderia bacterium.

Bacterial natural products have found many important industrial applications. Yet traditional discovery pipelines often prioritize individual natural product families despite the presence of multiple natural product biosynthetic gene clusters in each bacterial genome. Systematic characterization of talented strains is a means to expand the known natural product space. Here, we report genomics, epigenomics, and metabolomics studies of Burkholderia sp. FERM BP-3421, a soil isolate and known producer of antitumor spliceostatins. Its genome is composed of two chromosomes and two plasmids encoding at least 29 natural product families. Metabolomics studies showed that FERM BP-3421 also produces antifungal aminopyrrolnitrin and approved anticancer romidepsin. From the orphan metabolome features, we connected a lipopeptide of 1,928 Da to an 18-module nonribosomal peptide synthetase encoded as a single gene in chromosome 1. Isolation and structure elucidation led to the identification of selethramide which contains a repeating pattern of serine and leucine and is cyclized at the side chain oxygen of the one threonine residue at position 13. A (R)-3-hydroxybutyric acid moiety decorates the N-terminal serine. Initial attempts to obtain deletion mutants to probe the role of selethramide failed. After acquiring epigenome (methylome) data for FERM BP-3421, we employed a mimicry by methylation strategy that improved DNA transfer efficiency. Mutants defective in selethramide biosynthesis showed reduced surfactant activity and impaired swarming motility that could be chemically complemented with selethramide. This work unveils a lipopeptide that promotes surface motility, establishes improved DNA transfer efficiency, and sets the stage for continued natural product identification from a prolific strain.

Hypoxia controls the expression of genes responsible for serine synthesis in U87MG cells on ERN1-dependent manner.

Objective. Serine synthesis as well as endoplasmic reticulum stress and hypoxia are important factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of ERN1 (endoplasmic reticulum to nucleus signaling) significantly suppressed the glioblastoma cell proliferation and modified the hypoxia regulation. The present study is aimed to investigate the impact of hypoxia on the expression of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine aminotransferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) in U87MG glioblastoma cells in relation to knockdown of ERN1 with the intent to reveal the role of ERN1 signaling pathway on the endoplasmic reticulum stress-dependent regulation of expression of these genes. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed to hypoxia introduced by dimethyloxalylglycine for 4 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH, PSAT1, PDPH, SHMT1, and ATF4 genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that hypoxia up-regulated the expression level of PHGDH, PSAT1, and ATF4 genes in control U87MG cells, but PSPH and SHMT1 genes expression was down-regulated. The expression of PHGDH, PSAT1, and ATF4 genes in glioblastoma cells with knockdown of ERN1 signaling protein was more sensitive to hypoxia, especially PSAT1 gene. At the same time, the expression of PSPH gene in ERN1 knockdown cells was resistant to hypoxia. The expression of SHMT1 gene, encoding the enzyme responsible for conversion of serine to glycine, showed similar negative sensitivity to hypoxia in both control and ERN1 knockdown glioblastoma cells. Conclusion. The results of the present study demonstrate that the expression of genes responsible for serine synthesis is sensitive to hypoxia in gene-specific manner and that ERN1 knockdown significantly modifies the impact of hypoxia on the expression of PHGDH, PSAT1, PSPH, and ATF4 genes in glioblastoma cells and reflects the ERN1-mediated reprograming of hypoxic regulation at gene expression level.

Cytological and transcriptomic analysis to unveil the mechanism of web blotch resistance in Peanut.

BACKGROUND: Peanut is an important oil crop worldwide. Peanut web blotch is a fungal disease that often occurs at the same time as other leaf spot diseases, resulting in substantial leaf drop, which seriously affects the peanut yield and quality. However, the molecular mechanism underlying peanut resistance to web blotch is unknown. RESULTS: The cytological examination revealed no differences in the conidium germination rate between the web blotch-resistant variety ZH and the web blotch-susceptible variety PI at 12-48 hpi. The appressorium formation rate was significantly higher for PI than for ZH at 24 hpi. The papilla formation rate at 36 hpi and the hypersensitive response rate at 60 and 84 hpi were significantly higher for ZH than for PI. We also compared the transcriptional profiles of web blotch-infected ZH and PI plants at 0, 12, 24, 36, 48, 60, and 84 hpi using an RNA-seq technique. There were more differentially expressed genes (DEGs) in ZH and PI at 12, 36, 60, and 84 hpi than at 24 and 48 hpi. Moreover, there were more DEGs in PI than in ZH at each time-point. The analysis of metabolic pathways indicated that pantothenate and CoA biosynthesis; monobactam biosynthesis; cutin, suberine and wax biosynthesis; and ether lipid metabolism are specific to the active defense of ZH against YY187, whereas porphyrin metabolism as well as taurine and hypotaurine metabolism are pathways specifically involved in the passive defense of ZH against YY187. In the protein-protein interaction (PPI) network, most of the interacting proteins were serine acetyltransferases and cysteine synthases, which are involved in the cysteine synthesis pathway. The qRT-PCR data confirmed the reliability of the transcriptome analysis. CONCLUSION: On the basis of the PPI network for the significantly enriched genes in the pathways which were specifically enriched at different time points in ZH, we hypothesize that serine acetyltransferases and cysteine synthases are crucial for the cysteine-related resistance of peanut to web blotch. The study results provide reference material for future research on the mechanism mediating peanut web blotch resistance.