Plant serine/arginine-rich proteins: versatile players in RNA processing.

MAIN CONCLUSION: Serine/arginine-rich (SR) proteins participate in RNA processing by interacting with precursor mRNAs or other splicing factors to maintain plant growth and stress responses. Alternative splicing is an important mechanism involved in mRNA processing and regulation of gene expression at the posttranscriptional level, which is the main reason for the diversity of genes and proteins. The process of alternative splicing requires the participation of many specific splicing factors. The SR protein family is a splicing factor in eukaryotes. The vast majority of SR proteins' existence is an essential survival factor. Through its RS domain and other unique domains, SR proteins can interact with specific sequences of precursor mRNA or other splicing factors and cooperate to complete the correct selection of splicing sites or promote the formation of spliceosomes. They play essential roles in the composition and alternative splicing of precursor mRNAs, providing pivotal functions to maintain growth and stress responses in animals and plants. Although SR proteins have been identified in plants for three decades, their evolutionary trajectory, molecular function, and regulatory network remain largely unknown compared to their animal counterparts. This article reviews the current understanding of this gene family in eukaryotes and proposes potential key research priorities for future functional studies.

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

PERIOD (PER) and Casein Kinase 1δ regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. We found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

Mitotic CDK1 and 4E-BP1 I: Loss of 4E-BP1 serine 82 phosphorylation promotes proliferative polycystic disease and lymphoma in aged or sublethally irradiated mice.

4E-BP1 is a tumor suppressor regulating cap-dependent translation that is in turn controlled by mechanistic target of rapamycin (mTOR) or cyclin-dependent kinase 1 (CDK1) phosphorylation. 4E-BP1 serine 82 (S82) is phosphorylated by CDK1, but not mTOR, and the consequences of this mitosis-specific phosphorylation are unknown. Knock-in mice were generated with a single 4E-BP1 S82 alanine (S82A) substitution leaving other phosphorylation sites intact. S82A mice were fertile and exhibited no gross developmental or behavioral abnormalities, but the homozygotes developed diffuse and severe polycystic liver and kidney disease with aging, and lymphoid malignancies after irradiation. Sublethal irradiation caused immature T-cell lymphoma only in S82A mice while S82A homozygous mice have normal T-cell hematopoiesis before irradiation. Whole genome sequencing identified PTEN mutations in S82A lymphoma and impaired PTEN expression was verified in S82A lymphomas derived cell lines. Our study suggests that the absence of 4E-BP1S82 phosphorylation, a subtle change in 4E-BP1 phosphorylation, might predispose to polycystic proliferative disease and lymphoma under certain stressful circumstances, such as aging and irradiation.

Concentrations of vitamin B12 and folate in maternal serum and fetal fluids, metabolite interrelationships, and hepatic transcript abundance of key folate and methionine cycle genes: the impacts of maternal nutrition during the first 50 d of gestation.

Adequate maternal nutrition is key for proper fetal development and epigenetic programming. One-carbon metabolites (OCM), including vitamin B12, folate, choline, and methionine, play a role in epigenetic mechanisms associated with developmental programming. This study investigated the presence of B12 and folate in maternal serum, allantoic fluid (ALF), and amniotic fluid (AMF), as well as how those concentrations in all three fluids correlate to the concentrations of methionine-folate cycle intermediates in heifers receiving either a control (CON) or restricted (RES) diet for the first 50 d of gestation and fetal hepatic gene expression for methionine-folate cycle enzymes. Angus cross heifers (n = 43) were estrus synchronized, bred via artificial insemination with semen from a single sire, and randomly assigned to one of two nutrition treatments (CON = 20, RES = 23). Heifers were ovariohysterectomized on either day 16 (n = 14), 34 (n = 15), or 50 of gestation (n = 14), where samples of maternal serum (n = 42), ALF (n = 29), and AMF (n = 11) were collected and analyzed for concentrations of folate and B12. Concentrations of B12 and folate in ALF were greater (P < 0.05) in RES compared to CON. For ALF, folate concentrations were also greater (P < 0.01) on day 34 compared to day 50. There was a significant (P = 0.04) nutrition × fluid interaction for B12 concentrations where concentrations were greatest in restricted ALF, intermediate in control ALF, and lowest in CON and RES serum and AMF. Folate concentrations were greatest (P < 0.01) in ALF, intermediate in serum, and lowest in AMF. Additionally, positive correlations (P < 0.05) were found between ALF and AMF folate concentrations and AMF concentrations of methionine, serine, and glycine. Negative correlations (P < 0.05) between AMF folate and serum homocysteine were also observed. Both positive and negative correlations (P < 0.05) depending on the fluid evaluated were found between B12 and methionine, serine, and glycine concentrations. There was a downregulation (P = 0.05) of dihydrofolate reductase and upregulation (P = 0.03) of arginine methyltransferase 7 gene expression in RES fetal liver samples compared with CON fetal liver on day 50. Combined, these data show restricted maternal nutrition results in increased B12 and folate concentrations present in fetal fluids, and increased expression of genes for enzymes within one-carbon metabolism.

When pregnant cattle have restricted access to feed or specific nutrients, calf development can be affected, and the degree of impairment depends, at least partially, on timing, duration, and severity of the limitations. A biochemical pathway present in cells that can be affected by limited nutrition is one-carbon metabolism. This pathway is related to epigenetics, which regulates gene expression or the turning on and off of genes. Two important vitamins in one-carbon metabolism are vitamins B12 and folate. By understanding the amounts of those vitamins available to the developing calf, we can gain better insight into the regulation and potential avenues of improvement of calf growth and development. In this study, we found a nutrient restricted maternal diet increased the amount of B12 and folate in calf allantoic and amniotic fluids. We also found that folate and B12 were correlated to the presence of other nutrients in serum, allantoic fluid, and amniotic fluid. In addition, we found that a protein methylating gene in one-carbon metabolism had increased expression in calves from heifers receiving limited nutrition. This study is an important step in understanding how the nutrients available to a pregnant heifer during gestation affects nutrients available to the conceptus.

Analysis of Radiation Toxicity in Mammalian Cells Stably Transduced with Mitochondrial Stat3.

A coordinated action between nuclear and mitochondrial activities is essential for a proper cellular response to genotoxic stress. Several nuclear transcription factors, including STAT3, translocate to mitochondria to exert mitochondrial function regulation; however, the role of mitochondrial STAT3 (mitoSTAT3) under stressed conditions is still poorly understood. In this study, we examined whether the stable expression of mitoSTAT3 wild-type or mutated at the conserved serine residue (Ser727), which is involved in the mitochondrial function of STAT3, can affect the DNA damage response to UVC radiation. To address this issue, we generated mammalian cells (NIH-3T3 and HCT-116 cells) stably transduced to express the mitochondrial-targeted Stat3 gene in its wild-type or Ser727 mutated forms. Our results show that cell proliferation is enhanced in mitoStat3-transduced cells under both non-stressed and stressed conditions. Once irradiated with UVC, cells expressing wild-type mitoSTAT3 showed the highest cell survival, which was associated with a significant decrease in cell death. Low levels of oxidative stress were detected in UVC-irradiated NIH-3T3 cells expressing mitoSTAT3 wild-type or serine-related dominant active form (Ser727D), confirming a role of mitochondrial STAT3 in minimizing oxidant cellular stress that provides an advantage for cell survival.

Broad phosphorylation mediated by testis-specific serine/threonine kinases contributes to spermiogenesis and male fertility.

Genetic studies elucidate a link between testis-specific serine/threonine kinases (TSSKs) and male infertility in mammals, but the underlying mechanisms are unclear. Here, we identify a TSSK homolog in Drosophila, CG14305 (termed dTSSK), whose mutation impairs the histone-to-protamine transition during spermiogenesis and causes multiple phenotypic defects in nuclear shaping, DNA condensation, and flagellar organization in spermatids. Genetic analysis demonstrates that kinase catalytic activity of dTSSK, which is functionally conserved with human TSSKs, is essential for male fertility. Phosphoproteomics identify 828 phosphopeptides/449 proteins as potential substrates of dTSSK enriched primarily in microtubule-based processes, flagellar organization and mobility, and spermatid differentiation and development, suggesting that dTSSK phosphorylates various proteins to orchestrate postmeiotic spermiogenesis. Among them, the two substrates, protamine-like protein Mst77F/Ser9 and transition protein Mst33A/Ser237, are biochemically validated to be phosphorylated by dTSSK in vitro, and are genetically demonstrated to be involved in spermiogenesis in vivo. Collectively, our findings demonstrate that broad phosphorylation mediated by TSSKs plays an indispensable role in spermiogenesis.

Serine-arginine protein kinases and their targets in viral infection and their inhibition.

Accumulating evidence has consolidated the interaction between viral infection and host alternative splicing. Serine-arginine (SR) proteins are a class of highly conserved splicing factors critical for the spliceosome maturation, alternative splicing and RNA metabolism. Serine-arginine protein kinases (SRPKs) are important kinases that specifically phosphorylate SR proteins to regulate their distribution and activities in the central pre-mRNA splicing and other cellular processes. In addition to the predominant SR proteins, other cytoplasmic proteins containing a serine-arginine repeat domain, including viral proteins, have been identified as substrates of SRPKs. Viral infection triggers a myriad of cellular events in the host and it is therefore not surprising that viruses explore SRPKs-mediated phosphorylation as an important regulatory node in virus-host interactions. In this review, we briefly summarize the regulation and biological function of SRPKs, highlighting their involvement in the infection process of several viruses, such as viral replication, transcription and capsid assembly. In addition, we review the structure-function relationships of currently available inhibitors of SRPKs and discuss their putative use as antivirals against well-characterized viruses or newly emerging viruses. We also highlight the viral proteins and cellular substrates targeted by SRPKs as potential antiviral therapeutic candidates.

RACK1 release from the Ribosome Couples Translational Regulation with Starving Signaling and Possibly Depends on Phosphorylation of Key Serine and Threonine Residues.

The balance between protein anabolism and catabolism sets the foundations on which cells build their homeostasis. RACK1 is a ribosome-associated scaffold protein involved in signal transduction. On the ribosome, RACK1 enhances specific translation. Conversely, upon growth factor/nutrient starvation, RACK1 is present in a ribosome-free form and inhibits protein synthesis. However, the precise role of RACK1 when not bound to the ribosome still requires elucidation. Here, we show that extra-ribosomal RACK1 increases LC3-II accumulation, thereby mimicking an autophagy-like phenotype. Next, based on the ribosome-bound structure of RACK1, we suggest a possible mechanism for RACK1 release from the ribosome which relies on phosphorylation of precise amino acid residues, namely Thr39, Ser63, Thr86, Ser276, Thr277, Ser278, Ser279. Specifically, by performing an unbiased in silico screening using phospho-kinase prediction tools, we propose that, upon starving, AMPK1/2, ULK1/2 and PKR are the strongest candidate protein kinases to phosphorylate RACK1. This may be relevant in the context of caloric restriction and cancer therapy, where repressing translation of specific mRNAs would open important therapeutic avenues. Overall, our work provides novel insight into RACK1 function(s) by connecting its ribosomal and extra-ribosomal activities with translation and signaling.

Ultraviolet Photodissociation of Proteinogenic Amino Acids.

The ultraviolet photochemistry of the amino acids glycine, leucine, proline, and serine in their neutral forms was investigated using parahydrogen matrix-isolation spectroscopy. Irradiation by 213 nm light destroys the chirality of all three chiral amino acids as a result of the α-carbonyl C-C bond cleavage and hydrocarboxyl (HOCO) radical production. The temporal behavior of the Fourier-transform infrared spectra revealed that HOCO radicals rapidly reach a steady state, which occurs predominantly due to photodissociation of HOCO into CO + OH or CO2 + H. In glycine and leucine, the amine radicals generated by the α-carbonyl C-C bond cleavage rapidly undergo hydrogen elimination to yield methanimine and 3-methylbutane-1-imine, respectively. Breaking of the α-carbonyl C-C bond in proline appeared to yield 1-pyrroline, although due to its weak absorption it remains unconfirmed. In serine, additional products were formaldehyde and E/Z ethanimine. The present study shows that the direct production of HOCO previously observed in α-alanine generalizes to other amino acids of varying structure. It also revealed a tendency for amino acid photolysis to form imines rather than amine radicals. HOCO should be useful in the search for amino acids in interstellar space, particularly in combination with simple imine molecules.

A metabolome-wide Mendelian randomization study prioritizes potential causal circulating metabolites for multiple sclerosis.

To prioritize circulating metabolites that likely play causal roles in the pathogenesis of multiple sclerosis (MS). Two-sample Mendelian randomization analysis was performed to estimate the causal effects of 571 circulating metabolites on the risk of MS. Genetic instruments for circulating metabolites were obtained from three previous genome-wide association studies (GWAS) of the blood metabolome (N = 7824; 24,925; and 115,078; respectively), while genetic associations with MS were from a large GWAS by the International Multiple Sclerosis Genetics Consortium (14,802 cases and 26,703 control). The primary analysis was performed with the multiplicative random-effect inverse variance-weighted method, while multiple sensitivity analyses were conducted with the weighted median, weighted mode, MR-Egger, and MR-PRESSO. A total of 29 metabolites had suggestive evidence of causal associations with MS. Genetically instrumented levels of serine (OR = 1.56, 95% CI = 1.25-1.95), lysine (OR = 1.18, 95% CI = 1.01-1.38), acetone (OR = 2.45, 95% CI = 1.02-5.90), and acetoacetate (OR = 2.47, 95% CI = 1.14-5.34) were associated with a higher MS risk. Total cholesterol and phospholipids in large very-low-density lipoprotein were associated with a lower MS risk (OR = 0.83, 95% CI = 0.69-1.00; OR = 0.80, 95% CI = 0.68-0.95), but risk-increasing associations (OR = 1.20, 95% CI = 1.04-1.40; OR = 1.13, 95% CI = 1.00-1.28) were observed for the same two lipids in very large high-density lipoprotein. Our metabolome-wide Mendelian randomization study prioritized a list of circulating metabolites, such as serine, lysine, acetone, acetoacetate, and lipids, that likely have causal associations with MS.

Identification of the active ingredients and pharmacological effects of Kuntai capsules in the treatment of primary ovarian insufficiency: A review.

Kuntai capsules are effective in controlling primary ovarian insufficiency (POI). However, the precise mechanisms underlying the pharmacological effects of Kuntai capsules remain unclear. This study aimed to screen the active components and underlying mechanisms of Kuntai capsules for POI treatment using network pharmacology protocols and molecular docking technology. Potential active constituents in the chemical composition of Kuntai capsules were obtained from the Traditional Chinese Medicine System Pharmacology Database. Targets for POI were obtained from the Online Mendelian Inheritance in Man and Gene Cards database. All target data were integrated to identify the active ingredients of POI treatment. Enrichment analyses were performed using the Database for Annotation, Visualization, and Integrated Discovery database. The STRING database and Cytoscape software were used for protein-protein interaction network construction and core target identification. Finally, a molecular docking analysis of the active components and core targets was performed. A total of 157 ingredients related to POI were identified. Enrichment analysis showed that these components might participate in the mitogen-activated protein kinase, tumor necrosis factor, phosphoinositide-3-kinase/AKT serine/threonine kinase 1, and forkhead box O signaling pathways. Further protein-protein interaction network analysis revealed that the core targets were Jun proto-oncogene, AKT serine/threonine kinase 1, tumor protein P53, interleukin 6, and the epidermal growth factor receptor. Molecular docking analysis showed that baicalein was the most active ingredient with the highest affinity for the core targets. This study identified baicalein as the core functional component and elucidated the potential pharmacological effects of Kuntai capsule in the treatment of POI.

Heterologous production of the D-cycloserine intermediate O-acetyl-L-serine in a human type II pulmonary cell model.

Tuberculosis (TB) is the second leading cause of death by a single infectious disease behind COVID-19. Despite a century of effort, the current TB vaccine does not effectively prevent pulmonary TB, promote herd immunity, or prevent transmission. Therefore, alternative approaches are needed. We seek to develop a cell therapy that produces an effective antibiotic in response to TB infection. D-cycloserine (D-CS) is a second-line antibiotic for TB that inhibits bacterial cell wall synthesis. We have determined D-CS to be the optimal candidate for anti-TB cell therapy due to its effectiveness against TB, relatively short biosynthetic pathway, and its low-resistance incidence. The first committed step towards D-CS synthesis is catalyzed by the L-serine-O-acetyltransferase (DcsE) which converts L-serine and acetyl-CoA to O-acetyl-L-serine (L-OAS). To test if the D-CS pathway could be an effective prophylaxis for TB, we endeavored to express functional DcsE in A549 cells as a human pulmonary model. We observed DcsE-FLAG-GFP expression using fluorescence microscopy. DcsE purified from A549 cells catalyzed the synthesis of L-OAS as observed by HPLC-MS. Therefore, human cells synthesize functional DcsE capable of converting L-serine and acetyl-CoA to L-OAS demonstrating the first step towards D-CS production in human cells.

ASC Transporters Mediate D-Serine Transport into Astrocytes Adjacent to Synapses in the Mouse Brain.

D-serine is an important signalling molecule, which activates N-methyl D-aspartate receptors (NMDARs) in conjunction with its fellow co-agonist, the neurotransmitter glutamate. Despite its involvement in plasticity and memory related to excitatory synapses, its cellular source and sink remain a question. We hypothesise that astrocytes, a type of glial cell that surrounds synapses, are likely candidates to control the extracellular concentration of D-Serine by removing it from the synaptic space. Using in situ patch clamp recordings and pharmacological manipulation of astrocytes in the CA1 region of the mouse hippocampal brain slices, we investigated the transport of D-serine across the plasma membrane. We observed the D-serine-induced transport-associated currents upon puff-application of 10 mM D-serine on astrocytes. Further, O-benzyl-L-serine and trans-4-hydroxy-proline, known substrate inhibitors of the alanine serine cysteine transporters (ASCT), reduced D-serine uptake. These results indicate that ASCT is a central mediator of astrocytic D-serine transport and plays a role in regulating its synaptic concentration by sequestration into astrocytes. Similar results were observed in astrocytes of the somatosensory cortex and Bergmann glia in the cerebellum, indicative of a general mechanism expressed across a range of brain areas. This removal of synaptic D-serine and its subsequent metabolic degradation are expected to reduce its extracellular availability, influencing NMDAR activation and NMDAR-dependent synaptic plasticity.

Tetramate derivatives by chemoselective Dieckmann ring closure of allo-phenylserines, and their antibacterial activity.

A general route which provides direct access to substituted bicyclic tetramates, making use of Dieckmann cyclisation of oxazolidine derivatives derived from allo-phenylserines, is reported. Of interest is the high level of diastereoselectivity observed for the N-acylation reaction of oxazolidines and the complete chemoselectivity of their ring closure in the Dieckmann cyclisation. Significantly, the sense of the chemoselectivity is different to earlier reported threo-phenylserine systems, showing the importance of steric bulk around the bicyclic ring system. The derived C7-carboxamidotetramates, but not C7-acyl systems, exhibited potent antibacterial activity against MRSA, with the most active compounds exhibiting well-defined physicochemical and structure-activity properties. This work clearly demonstrates that densely functionalised tetramates are both readily available and may exhibit high levels of antibacterial activity.

Standardized ileal digestibility of amino acids in cottonseed meal fed to pregnant and non-pregnant sows.

This study determined the apparent ileal digestibility (AID) and standard ileal digestibility (SID) of crude protein (CP) and amino acids (AA) of six cottonseed meal (CSM) samples in pregnant and non-pregnant sows. Two CSM samples were processed by expelling with a CP level of 40.67% (ECSM41) and 44.64% (ECSM45), and four samples were processed by solvent-extracted which contained graded CP levels of 45% (SECSM45), 51.16% (SECSM51), 56.44% (SECSM56), and 59.63% (SECSM60). Landrace ×Yorkshire third parity sows, 7 at gestation and 14 non-pregnant, were fitted with T-cannula in the distal ileum. Pregnant sows were allotted to a 7 × 6 Latin square design with a cornstarch-based nitrogen-free (NF) diet and the six CSM diets, and non-pregnant sows were allotted to a replicated 7 × 3 Latin square design with seven diets and three periods, respectively, resulting in a total of six replicates per treatment. All experimental sows were fed 3.0 kg/d of the experimental diets. The AID of CP in ECSM41 (75.58%) was lower than in SECSM51 (80.42%), SECSM56 (80.50%), and SECSM60 (82.44%) diets for pregnant sows (P < 0.05). The AID of CP in ECSM41 (77.88%) was significantly lower than in SECSM60 (81.87%) diets for non-pregnant sows (P < 0.05). The physiological phase did not affect the AID of CP (P > 0.05). The SID of CP was affected by diets for both pregnant (P < 0.01) and non-pregnant sows (P = 0.06). The physiological phase also affected the SID of CP (P < 0.01). The AID of histidine, leucine, methionine, threonine, and tryptophan significantly differed between different CSM samples in both pregnant (P < 0.05) and non-pregnant sows (P < 0.05). The AID of dispensable AA aspartic acid, cysteine, glutamic acid, serine, and tyrosine differed between different CSM samples of both pregnant (P < 0.05) and non-pregnant sows (P < 0.05). For pregnant sows, the indispensable AA cysteine, glycine, proline, and tyrosine had significantly different SID between different groups (P < 0.05). For non-pregnant sows, the SID of arginine, lysine, methionine, threonine, aspartic acid, cysteine, and serine had different values among different diets (P < 0.05). In conclusion, the current study presented that the ileal AA digestibility of CSM fed to pregnant and non-pregnant sows increased with the decreased of fiber content, and the current findings can contribute to a precise formulation of diets for sows using CSM.

As a protein-rich cottonseed byproduct, cottonseed meal (CSM) is considered a vegetable protein source that can substitute soybean meal in the feed of livestock animals. However, the presence of free gossypol and high fiber levels in CSM have been limiting factors for its use in growing and finishing pigs, yet its nutritive value is still uncertain for sows. There is a lack of standard ileal digestibility (SID) of amino acids (AA) for plant proteins because fitting a T-cannula in the distal ileum is difficult. Therefore, this study evaluated the apparent ileal digestibility and SID of 18 AA of CSM in sows at two physiological stages (gestation and non-pregnancy). We found that CSM with different chemical compositions impacted the SID of AA when fed to pregnant and non-pregnant sows. Additionally, the physiological stage of the sow has a substantial impact on the SID of some AA. The current findings of this study provided a basis for the precise formulation of sow diets with CSM.

Vemurafenib improves muscle histopathology in a mouse model of LAMA2-related congenital muscular dystrophy.

Laminin-α2-related congenital muscular dystrophy (LAMA2-CMD) is a neuromuscular disease affecting around 1-9 in 1,000,000 children. LAMA2-CMD is caused by mutations in the LAMA2 gene resulting in the loss of laminin-211/221 heterotrimers in skeletal muscle. LAMA2-CMD patients exhibit severe hypotonia and progressive muscle weakness. Currently, there is no effective treatment for LAMA2-CMD and patients die prematurely. The loss of laminin-α2 results in muscle degeneration, defective muscle repair and dysregulation of multiple signaling pathways. Signaling pathways that regulate muscle metabolism, survival and fibrosis have been shown to be dysregulated in LAMA2-CMD. As vemurafenib is a US Food and Drug Administration (FDA)-approved serine/threonine kinase inhibitor, we investigated whether vemurafenib could restore some of the serine/threonine kinase-related signaling pathways and prevent disease progression in the dyW-/- mouse model of LAMA2-CMD. Our results show that vemurafenib reduced muscle fibrosis, increased myofiber size and reduced the percentage of fibers with centrally located nuclei in dyW-/- mouse hindlimbs. These studies show that treatment with vemurafenib restored the TGF-ß/SMAD3 and mTORC1/p70S6K signaling pathways in skeletal muscle. Together, our results indicate that vemurafenib partially improves histopathology but does not improve muscle function in a mouse model of LAMA2-CMD.

Specific Deoxyceramide Species Correlate with Expression of Macular Telangiectasia Type 2 (MacTel2) in a SPTLC2 Carrier HSAN1 Family.

Hereditary sensory and autonomic neuropathy type 1 (HSAN1/HSN1) is a peripheral neuropathy most commonly associated with pathogenic variants in the serine palmitoyltransferase complex (SPTLC1, SPTLC2) genes, which are responsible for sphingolipid biosynthesis. Recent reports have shown that some HSAN1 patients also develop macular telangiectasia type 2 (MacTel2), a retinal neurodegeneration with an enigmatic pathogenesis and complex heritability. Here, we report a novel association of a SPTLC2 c.529A>G p.(Asn177Asp) variant with MacTel2 in a single member of a family that otherwise has multiple members afflicted with HSAN1. We provide correlative data to suggest that the variable penetrance of the HSAN1/MacTel2-overlap phenotype in the proband may be explained by levels of certain deoxyceramide species, which are aberrant intermediates of sphingolipid metabolism. We provide detailed retinal imaging of the proband and his HSAN1+/MacTel2- brothers and suggest mechanisms by which deoxyceramide levels may induce retinal degeneration. This is the first report of HSAN1 vs. HSAN1/MacTel2 overlap patients to comprehensively profile sphingolipid intermediates. The biochemical data here may help shed light on the pathoetiology and molecular mechanisms of MacTel2.

Synthesis of Disulfide Surrogate Peptides through a Serine/Threonine Ligation-Assisted Diaminodiacid Strategy.

We report a new serine/threonine ligation (STL)-assisted diaminodiacid (DADA) strategy for the flexible construction of disulfide surrogates by the option of more abundant -Aa-Ser/Thr- ligation sites. The practicality of this strategy was evidenced by the synthesis of the intrachain disulfide surrogate of C-type natriuretic peptide and the interchain disulfide surrogate of insulin.

iPSC-derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function.

Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations and GWAS have linked MacTel to decreased circulating levels of serine and elevated levels of neurotoxic 1-deoxysphingolipids (1-dSLs). However, retina-specific, disease-contributing factors have yet to be identified. Here, we used iPSC-differentiated retinal pigmented epithelial (iRPE) cells derived from donors with or without MacTel to screen for novel cell-intrinsic pathological mechanisms. We show that MacTel iRPE cells mimicked the low serine levels observed in serum from patients with MacTel. Through RNA-Seq and gene set enrichment pathway analysis, we determined that MacTel iRPE cells are enriched in cellular stress pathways and dysregulation of central carbon metabolism. Using respirometry and mitochondrial stress testing, we functionally validated that MacTel iRPE cells had a reduction in mitochondrial function that was independent of defects in serine biosynthesis and 1-dSL accumulation. Thus, we identified phenotypes that may constitute alternative disease mechanisms beyond the known serine/sphingolipid pathway.

The Acute Effects and Mechanism of Ketamine on Nicotine-Induced Neurogenic Relaxation of the Corpus Cavernosum in Mice.

The present study aimed to investigate the acute effects and the mechanism of ketamine on nicotine-induced relaxation of the corpus cavernosum (CC) in mice. This study measured the intra-cavernosal pressure (ICP) of male C57BL/6 mice and the CC muscle activities using an organ bath wire myograph. Various drugs were used to investigate the mechanism of ketamine on nicotine-induced relaxation. Direct ketamine injection into the major pelvic ganglion (MPG) inhibited MPG-induced increases in ICP. D-serine/L-glutamate-induced relaxation of the CC was inhibited by MK-801 (N-methyl-D-aspartate (NMDA) receptor inhibitor), and nicotine-induced relaxation was enhanced by D-serine/L-glutamate. NMDA had no effect on CC relaxation. Nicotine-induced relaxation of the CC was suppressed by mecamylamine (a non-selective nicotinic acetylcholine receptor antagonist), lidocaine, guanethidine (an adrenergic neuronal blocker), Nw-nitro-L-arginine (a non-selective nitric oxide synthase inhibitor), MK-801, and ketamine. This relaxation was almost completely inhibited in CC strips pretreated with 6-hydroxydopamine (a neurotoxic synthetic organic compound). Ketamine inhibited cavernosal nerve neurotransmission via direct action on the ganglion and impaired nicotine-induced CC relaxation. The relaxation of the CC was dependent on the interaction of the sympathetic and parasympathetic nerves, which may be mediated by the NMDA receptor.