Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD.
- MeSH
- cilie patologie MeSH
- kationtové kanály TRPP genetika metabolismus MeSH
- kinasy NEK genetika metabolismus MeSH
- ledviny metabolismus MeSH
- lidé MeSH
- mutace MeSH
- myši MeSH
- novorozenec MeSH
- polycystická choroba ledvin * genetika MeSH
- polycystické ledviny autozomálně dominantní * patologie MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- serin genetika metabolismus MeSH
- transportní proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- novorozenec MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The most frequent alterations in plasma amino acid concentrations in type 1 and type 2 diabetes are decreased L-serine and increased branched-chain amino acid (BCAA; valine, leucine, and isoleucine) levels. The likely cause of L-serine deficiency is decreased synthesis of 3-phosphoglycerate, the main endogenous precursor of L-serine, due to impaired glycolysis. The BCAA levels increase due to decreased supply of pyruvate and oxaloacetate from glycolysis, enhanced supply of NADH + H+ from beta-oxidation, and subsequent decrease in the flux through the citric acid cycle in muscles. These alterations decrease the supply of α-ketoglutarate for BCAA transamination and the activity of branched-chain keto acid dehydrogenase, the rate-limiting enzyme in BCAA catabolism. L-serine deficiency contributes to decreased synthesis of phospholipids and increased synthesis of deoxysphinganines, which play a role in diabetic neuropathy, impaired homocysteine disposal, and glycine deficiency. Enhanced BCAA levels contribute to increased levels of aromatic amino acids (phenylalanine, tyrosine, and tryptophan), insulin resistance, and accumulation of various metabolites, whose influence on diabetes progression is not clear. It is concluded that amino acid concentrations should be monitored in patients with diabetes, and systematic investigation is needed to examine the effects of L-serine and glycine supplementation on diabetes progression when these amino acids are decreased.
Decreasing the dietary intake of methionine exerts robust anti-adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), that is, sulfur amino acid restriction (SAAR). Contrarily, SAAR diets formulated for human consumption included cysteine, and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not methionine, suggesting that CR, but not MR, is responsible for the anti-adiposity effects of SAAR. Whether this is true, and, if so, the underlying mechanisms are unknown. Using methionine- and cysteine-titrated diets, we demonstrate that the anti-adiposity effects of SAAR are due to CR. Data indicate that CR increases serinogenesis (serine biosynthesis from non-glucose substrates) by diverting substrates from glyceroneogenesis, which is essential for fatty acid reesterification and triglyceride synthesis. Molecular data suggest that CR depletes hepatic glutathione and induces Nrf2 and its downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck-M. In mice, the magnitude of SAAR-induced changes in molecular markers depended on dietary fat concentration (60% fat >10% fat), sex (males > females), and age-at-onset (young > adult). Our findings are translationally relevant as we found negative and positive correlations of plasma serine and cysteine, respectively, with triglycerides and metabolic syndrome criteria in a cross-sectional epidemiological study. Controlled feeding of low-SAA, high-polyunsaturated fatty acid diets increased plasma serine in humans. Serinogenesis might be a target for treating hypertriglyceridemia.
- MeSH
- aminokyseliny sírové * metabolismus MeSH
- cystein * metabolismus MeSH
- lidé MeSH
- metabolismus lipidů MeSH
- methionin metabolismus MeSH
- myši MeSH
- obezita metabolismus MeSH
- průřezové studie MeSH
- serin metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Signal transducer and activator of transcription 3 (Stat3) is responsible for many aspects of normal development and contributes to the development and progression of cancer through regulating epithelial cell identity and cancer stem cells. In breast cancer, Stat3 is associated with triple-negative breast cancers (TNBC) and its function has been related to the activation of p63, itself a marker of basal-like TNBC and a master regulator of stem cell activities. Stat3 activation is controlled by dual phosphorylation at tyrosine 705 (pTyr705) and serine 727 (pSer727), although it is unclear whether these have equivalent effects, and whether they are related or independent events. To address these issues, we investigated Stat3 phosphorylation at the two sites by immunohistochemistry in 173 patients with TNBC. Stat3 phosphorylation was assessed by automated quantitative measurements of digitized scanned images and classified into four categories based on histoscore. The results were analyzed for associations with multiple markers of tumor phenotype, proliferation, BRCA status, and clinicopathological characteristics. We show that the levels of pTyr705- and pSer727-Stat3 were independent in 34% of tumors. High pTyr705-Stat3 levels were associated with the luminal differentiation markers ERβ/AR and MUC1, whereas tumors with high levels of pSer727-Stat3 were more likely to be positive for the basal marker CK5/6, but were independent of p63 and were EGFR negative. Combined high pSer727- and low Tyr705-Stat3 phosphorylation associated with basal-like cancer. Although high Stat3 phosphorylation levels were associated with less aggressive tumor characteristics, they did not associate with improved survival, indicating that Stat3 phosphorylation is an unfavorable indicator for tumors with an otherwise good prognosis according to clinicopathological characteristics. These findings also show that pTyr705-Stat3 and pSer727-Stat3 associate with specific breast tumor phenotypes, implying that they exert distinct functional activities in breast cancer.
- MeSH
- fenotyp MeSH
- fosforylace MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- serin genetika MeSH
- transkripční faktor STAT3 metabolismus MeSH
- triple-negativní karcinom prsu * patologie MeSH
- tyrosin genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
HDMX and its homologue HDM2 are two essential proteins for the cell; after genotoxic stress, both are phosphorylated near to their RING domain, specifically at serine 403 and 395, respectively. Once phosphorylated, both can bind the p53 mRNA and enhance its translation; however, both recognize p53 protein and provoke its degradation under normal conditions. HDM2 has been well-recognized as an E3 ubiquitin ligase, whereas it has been reported that even with the high similarity between the RING domains of the two homologs, HDMX does not have the E3 ligase activity. Despite this, HDMX is needed for the proper p53 poly-ubiquitination. Phosphorylation at serine 395 changes the conformation of HDM2, helping to explain the switch in its activity, but no information on HDMX has been published. Here, we study the conformation of HDMX and its phospho-mimetic mutant S403D, investigate its E3 ligase activity and dissect its binding with p53. We show that phospho-mutation does not change the conformation of the protein, but HDMX is indeed an E3 ubiquitin ligase in vitro; however, in vivo, no activity was found. We speculated that HDMX is regulated by induced fit, being able to switch activity accordingly to the specific partner as p53 protein, p53 mRNA or HDM2. Our results aim to contribute to the elucidation of the contribution of the HDMX to p53 regulation.
- MeSH
- jaderné proteiny genetika MeSH
- messenger RNA metabolismus MeSH
- nádorový supresorový protein p53 * genetika metabolismus MeSH
- proteiny buněčného cyklu metabolismus MeSH
- protoonkogenní proteiny c-mdm2 * genetika metabolismus MeSH
- protoonkogenní proteiny genetika MeSH
- serin metabolismus MeSH
- ubikvitin genetika MeSH
- ubikvitinace MeSH
- ubikvitinligasy genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Campylobacter (C.) spp. represent one of the most important causes for food-borne bacterial pathogen in humans worldwide. The aim of this study was to investigate metabolic requirements of two Campylobacter strains of different species based on substrate utilisation (in vitro). Based on these results, a correlation between the colonisation and the available substrates in different intestinal sections was recorded using an animal model. Campylobacter coli (ST-5777) and C. jejuni (ST-122) were used to inoculate 16 pigs, respectively, and one group of 16 pigs was used as control. The strains differed significantly in substrate utilisation - C. coli was able to metabolise various substrates (acetate, asparagine, serine, fucose, and propionate), while C. jejuni only utilised serine. Metabolomic analysis of intestinal content from different gut sections showed the presence of all previously tested metabolites, except for fucose. A significantly larger amount of glucose was found in the jejunum of those pigs infected with C. coli, while neither strain utilised it in vitro. The analysis of the intestinal contents revealed a very low proportion of Campylobacterales in the total microbiome, suggesting that the small percentage of the inoculated Campylobacter strains in the gut microflora of the animals is too low to cause differences between the control and infected groups in the composition of the metabolome. Nevertheless, knowledge of specific nutritional requirements of the pathogens combined with proof of different metabolites in the intestinal segments may provide clues about the site of colonisation in the host and improve our understanding of this zoonotic germ.
- MeSH
- Campylobacter coli * MeSH
- Campylobacter jejuni * MeSH
- Campylobacter * MeSH
- fukosa MeSH
- kampylobakterové infekce * mikrobiologie veterinární MeSH
- lidé MeSH
- nemoci prasat * MeSH
- prasata MeSH
- serin MeSH
- střeva MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Generation of functional gametes is accomplished through a multilayered and finely orchestrated succession of events during meiotic progression. In the Caenorhabditis elegans germline, the HORMA-domain-containing protein HTP-3 plays pivotal roles for the establishment of chromosome axes and the efficient induction of programmed DNA double-strand breaks, both of which are crucial for crossover formation. Double-strand breaks allow for accurate chromosome segregation during the first meiotic division and therefore are an essential requirement for the production of healthy gametes. Phosphorylation-dependent regulation of HORMAD protein plays important roles in controlling meiotic chromosome behavior. Here, we document a phospho-site in HTP-3 at Serine 285 that is constitutively phosphorylated during meiotic prophase I. pHTP-3S285 localization overlaps with panHTP-3 except in nuclei undergoing physiological apoptosis, in which pHTP-3 is absent. Surprisingly, we observed that phosphorylation of HTP-3 at S285 is independent of the canonical kinases that control meiotic progression in nematodes. During meiosis, the htp-3(S285A) mutant displays accelerated RAD-51 turnover, but no other meiotic abnormalities. Altogether, these data indicate that the Ser285 phosphorylation is independent of canonical meiotic protein kinases and does not regulate HTP-3-dependent meiotic processes. We propose a model wherein phosphorylation of HTP-3 occurs through noncanonical or redundant meiotic kinases and/or is likely redundant with additional phospho-sites for function in vivo.
- MeSH
- Caenorhabditis elegans genetika metabolismus MeSH
- fosforylace MeSH
- meióza * MeSH
- proteiny buněčného cyklu genetika MeSH
- proteiny Caenorhabditis elegans * metabolismus MeSH
- segregace chromozomů MeSH
- serin metabolismus MeSH
- synaptonemální komplex metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
L-serine plays an essential role in a broad range of cellular functions including protein synthesis, neurotransmission, and folate and methionine cycles and synthesis of sphingolipids, phospholipids, and sulphur containing amino acids. A hydroxyl side-chain of L-serine contributes to polarity of proteins, and serves as a primary site for binding a phosphate group to regulate protein function. D-serine, its D-isoform, has a unique role. Recent studies indicate increased requirements for L-serine and its potential therapeutic use in some diseases. L-serine deficiency is associated with impaired function of the nervous system, primarily due to abnormal metabolism of phospholipids and sphingolipids, particularly increased synthesis of deoxysphingolipids. Therapeutic benefits of L-serine have been reported in primary disorders of serine metabolism, diabetic neuropathy, hyperhomocysteinemia, and amyotrophic lateral sclerosis. Use of L-serine and its metabolic products, specifically D-serine and phosphatidylserine, has been investigated for the therapy of renal diseases, central nervous system injury, and in a wide range of neurological and psychiatric disorders. It is concluded that there are disorders in which humans cannot synthesize L-serine in sufficient quantities, that L-serine is effective in therapy of disorders associated with its deficiency, and that L-serine should be classified as a "conditionally essential" amino acid.
- MeSH
- esenciální aminokyseliny MeSH
- fosfolipidy MeSH
- lidé MeSH
- serin * MeSH
- sfingolipidy metabolismus MeSH
- vrozené poruchy metabolismu aminokyselin * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Astrocytes are the most abundant cell type in the human brain and are important regulators of several critical cellular functions, including synaptic transmission. Although astrocytes are known to play a central role in the etiology and pathophysiology of schizophrenia, little is known about their potential involvement in clinical response to the antipsychotic clozapine. Moreover, astrocytes display a remarkable degree of morphological diversity, but the potential contribution of astrocytic subtypes to disease biology and drug response has received little attention. Here, we used state-of-the-art human induced pluripotent stem cell (hiPSC) technology to derive a morphological subtype of astrocytes from healthy individuals and individuals with schizophrenia, including responders and nonresponders to clozapine. Using functional assays and transcriptional profiling, we identified a distinct gene expression signature highly specific to schizophrenia as shown by disease association analysis of more than 10 000 diseases. We further found reduced levels of both glutamate and the NMDA receptor coagonist d-serine in subtype astrocytes derived from schizophrenia patients, and that exposure to clozapine only rescued this deficiency in cells from clozapine responders, providing further evidence that d-serine in particular, and NMDA receptor-mediated glutamatergic neurotransmission in general, could play an important role in disease pathophysiology and clozapine action. Our study represents a first attempt to explore the potential contribution of astrocyte diversity to schizophrenia pathophysiology using a human cellular model. Our findings suggest that specialized subtypes of astrocytes could be important modulators of disease pathophysiology and clinical drug response, and warrant further investigations.
- MeSH
- antipsychotika farmakologie MeSH
- astrocyty metabolismus MeSH
- dospělí MeSH
- indukované pluripotentní kmenové buňky MeSH
- klozapin farmakologie MeSH
- kyselina glutamová metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- schizofrenie farmakoterapie metabolismus MeSH
- serin metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
