Serine-threonine Research Articles

The serine-threonine protein kinase Snf1 orchestrates the expression of plant cell wall-degrading enzymes and is required for full virulence of the maize pathogen Colletotrichum graminicola

Colletotrichum graminicola, the causal agent of maize leaf anthracnose and stalk rot, differentiates a pressurized infection cell called an appressorium in order to invade the epidermal cell, and subsequently forms biotrophic and necrotrophic hyphae to colonize the host tissue. While the role of force in appressorial penetration is established (Bechinger et al., 1999), the involvement of cell wall-degrading enzymes (CWDEs) in this process and in tissue colonization is poorly understood, due to the enormous number and functional redundancy of these enzymes. The serine/threonine protein kinase gene SNF1 identified in Sucrose Non-Fermenting yeast mutants mediates de-repression of catabolite-repressed genes, including many genes encoding CWDEs. In this study, we identified and functionally characterized the SNF1 homolog of C. graminicola. Δsnf1 mutants showed reduced vegetative growth and asexual sporulation rates on media containing polymeric carbon sources. Microscopy revealed reduced efficacies in appressorial penetration of cuticle and epidermal cell wall, and formation of unusual medusa-like biotrophic hyphae by Δsnf1 mutants. Severe and moderate virulence reductions were observed on intact and wounded leaves, respectively. Employing RNA-sequencing we show for the first time that more than 2,500 genes are directly or indirectly controlled by Snf1 in necrotrophic hyphae of a plant pathogenic fungus, many of which encode xylan- and cellulose-degrading enzymes. The data presented show that Snf1 is a global regulator of gene expression and is required for full virulence.

Deciphering the features and functions of serine/arginine protein kinases in bread wheat

Serine/arginine protein kinases (SRPKs) are members of the serine-threonine kinase family that phosphorylate the Serine/arginine-rich (SR) proteins involved in alternate splicing. They are reported in various eukaryotes including mammals, and in a few plants, but seldom explored in important crop species. Herein, we identified a total of nine TaSRPK genes from all three subgenomes (A, B, and D) of a staple crop Triticum aestivum, and phylogenetically classified them into two groups. The TaSRPKs have conserved gene architecture with four exons. Each TaSRPK protein consists of a characteristic protein kinase domain having an active site and ATP binding region. The occurrence of diverse cis-regulatory elements in the promoter region, and interaction with assorted groups of transcription factors and miRNAs exhibited their divergent functions. Differential expression of certain TaSRPKs in vegetative and reproductive tissues and in the presence of fungal pathogens and various abiotic stress conditions further assured their association during development processes and stress response. Our study highlighted the importance of TaSRPKs, which might be useful for their detailed characterization in future research.

Loss of VRK1 alters the nuclear phosphoproteome in the DNA damage response to doxorubicin

Dynamic chromatin remodeling requires regulatory mechanisms for its adaptation to different nuclear function, which are likely to be mediated by signaling proteins. In this context, VRK1 is a nuclear Ser-Thr kinase that regulates pathways associated with transcription, replication, recombination, and DNA repair. Therefore, VRK1 is a potential regulatory, or coordinator, molecule in these processes. In this work we studied the effect that VRK1 depletion has on the basal nuclear and chromatin phosphoproteome, and their associated pathways. VRK1 depletion caused an alteration in the pattern of the nuclear phosphoproteome, which is mainly associated with nucleoproteins, ribonucleoproteins, RNA splicing and processing. Next, it was determined the changes in proteins associated with DNA damage that was induced by doxorubicin treatment. Doxorubicin alters the nuclear phosphoproteome affecting proteins implicated in DDR, including DSB repair proteins NBN and 53BP1, cellular response to stress and chromatin organization proteins. In VRK1-depleted cells, the effect of doxorubicin on protein phosphorylation was reverted to basal levels. The nuclear phosphoproteome patterns induced by doxorubicin are altered by VRK1 depletion, and is enriched in histone modification proteins and chromatin associated proteins. These results indicate that VRK1 plays a major role in processes requiring chromatin remodeling in its adaptation to different biological contexts.

M6A-Mediated Upregulation of Imprinted in Prader-Willi Syndrome Induces Aberrant Apical-Basal Polarization and Oxidative Damage in RPE Cells.

To reveal the clinical significance, pathological involvement and molecular mechanism of imprinted in Prader-Willi syndrome (IPW) in RPE anomalies that contribute to AMD. IPW expression under pathological conditions were detected by microarrays and qPCR assays. In vitro cultured fetal RPE cells were used to study the pathogenicity induced by IPW overexpression and to analyze its upstream and downstream regulatory networks. We showed that IPW is upregulated in the macular RPE-choroid tissue of dry AMD patients and in fetal RPE cells under oxidative stress, inflammation and dedifferentiation. IPW overexpression in fetal RPE cells induced aberrant apical-basal polarization as shown by dysregulated polarized markers, disrupted tight and adherens junctions, and inhibited phagocytosis. IPW upregulation was also associated with RPE oxidative damages, as demonstrated by intracellular accumulation of reactive oxygen species, reduced cell proliferation, and accelerated cell apoptosis. Mechanically, N6-methyladenosine level of the IPW transcript regulated its stability with YTHDC1 as the reader. IPW mediated RPE features by suppressing MEG3 expression to sequester its inhibition on the AKT serine-threonine kinase (AKT)/mammalian target of rapamycin (mTOR) pathway. We also noticed that the mTOR inhibitor rapamycin suppresses the AKT/mTOR pathway to alleviate the IPW-induced RPE anomalies. We revealed that IPW overexpression in RPE induces aberrant apical-basal polarization and oxidative damages, thus contributing to AMD progression. We also annotated the upstream and downstream regulatory networks of IPW in RPE. Our findings shed new light on the molecular mechanisms of RPE dysfunctions, and indicate that IPW blockers may be a promising option to treat RPE abnormalities in AMD.

Serine/Threonine Kinase (STK) 33 promotes the proliferation and metastasis of human esophageal squamous cell carcinoma via inflammation-related pathway

The serine/threonine kinase (STK) 33 plays a key role in cancer cell proliferation and metastasis. Abnormal STK33 expression is closely related to malignancy of numerous cancers. This study suggests the important role of STK33 in the pathogenesis and metastatic progression of esophageal squamous cell carcinoma (ESCC). STK33 expression in human ESCC tissues was detected by immunohistochemical technique. Further, we analyzed the relationship between STK33 and clinical and pathological factors as well as the prognosis of patients. ECa109 cell line was cultured and transfected with STK33-RNAi lentiviral vector to perform Hochest33342 & PI and metastasis experiments. The TCGA database was used to analyze the STK33 expression level in ESCC. All statistical analyses were performed in SPSS 23.0 software. Differences with P < 0.05 were considered statistically significant. In human ESCC specimens, STK33 was overexpressed and associated with poor prognosis. Silencing STK33 expression suppressed ESCC proliferation, migration, invasion, and tumor growth. STK33 also mediated angiogenesis, TGFβ, and inflammatory response in ESCC. Mechanistic investigations revealed that STK33 regulates ESCC through multiple complex pathways. Dysregulated STK33 signaling promotes ESCC growth and progression. Thus, our findings identified STK33 as a candidate treatment target that improves ESCC therapy.

Protein Kinase STK24 Promotes Tumor Immune Evasion via the AKT-PD-L1 Axis.

Immunotherapy targeting PD-L1 is still ineffective for a wide variety of tumors with high unpredictability. Deploying combined immunotherapy with alternative targeting is practical to overcome this therapeutic resistance. Here, the deficiency of serine-threonine kinase STK24 is observed in tumor cells causing substantial attenuation of tumor growth in murine syngeneic models, a process relying on cytotoxic CD8+ T and NK cells. Mechanistically, STK24 in tumor cells associates with and directly phosphorylates AKT at Thr21, which promotes AKT activation and subsequent PD-L1 induction. Deletion or inhibition of STK24, by contrast, blocks IFN-γ-mediated PD-L1 expression. Various murine models indicate that in vivo silencing of STK24 can significantly enhance the efficacy of the anti-PD-1 blockade strategy. Elevated STK24 levels are observed in patient specimens in multiple tumor types and inversely correlated with intratumoral infiltration of cytotoxic CD8+ T cells and with patient survival. The study collectively identifies STK24 as a critical modulator of antitumor immunity, which engages in AKT and PD-L1/PD-1 signaling and is a promising target for combined immunotherapy.

Evaluation of Tyrosine Kinase Inhibitors Loaded Injectable Hydrogels for Improving Connexin43 Gap Junction Intercellular Communication.

Myocardial infarction (MI) is one of the leading causes of death in the developed world, and the loss of cardiomyocytes plays a critical role in the pathogenesis of heart failure. Implicated in this process is a decrease in gap junction intercellular communication due to remodeling of Connexin43 (Cx43). We previously identified that intraperitoneal injection of the Pyk2 inhibitor PF4618433 reduced infarct size, maintained Cx43 at the intercalated disc in left ventricle hypertrophic myocytes, and improved cardiac function in an MI animal model of heart failure. With the emergence of injectable hydrogels as a therapeutic toward the regeneration of cardiac tissue after MI, here, we provide proof of concept that the release of tyrosine kinase inhibitors from hydrogels could have beneficial effects on cardiomyocytes. We developed an injectable hydrogel consisting of thiolated hyaluronic acid and P123-maleimide micelles that can incorporate PF4618433 as well as the Src inhibitor Saracatinib and achieved sustained release (of note, Src activates Pyk2). Using neonatal rat ventricular myocytes in the presence of a phorbol ester, endothelin-1, or phenylephrine to stimulate cardiac hypertrophy, the release of PF4618433 from the hydrogel had the same ability to decrease Cx43 tyrosine phosphorylation and maintain Cx43 localization at the plasma membrane as when directly added to the growth media. Additional beneficial effects included decreases in apoptosis, the hypertrophic marker atrial natriuretic peptide (ANP), and serine kinases upregulated in hypertrophy. Finally, the presence of both PF4618433 and Saracatinib further decreased the level of ANP and apoptosis than each inhibitor alone, suggesting that a combinatorial approach may be most beneficial. These findings provide the groundwork to test if tyrosine kinase inhibitor release from hydrogels will have a beneficial effect in an animal model of MI-induced heart failure.

The induction of SHP-1 degradation by TAOK3 ensures the responsiveness of T cells to TCR stimulation.

Thousand-and-one-amino acid kinase 3 (TAOK3) is a serine and threonine kinase that belongs to the STE-20 family of kinases. Its absence reduces T cell receptor (TCR) signaling and increases the interaction of the tyrosine phosphatase SHP-1, a major negative regulator of proximal TCR signaling, with the kinase LCK, a component of the core TCR signaling complex. Here, we used mouse models and human cell lines to investigate the mechanism by which TAOK3 limits the interaction of SHP-1 with LCK. The loss of TAOK3 decreased the survival of naïve CD4+ T cells by dampening the transmission of tonic and ligand-dependent TCR signaling. In mouse T cells, Taok3 promoted the secretion of interleukin-2 (IL-2) in response to TCR activation in a manner that depended on Taok3 gene dosage and on Taok3 kinase activity. TCR desensitization in Taok3-/- T cells was caused by an increased abundance of Shp-1, and pharmacological inhibition of Shp-1 rescued the activation potential of these T cells. TAOK3 phosphorylated threonine-394 in the phosphatase domain of SHP-1, which promoted its ubiquitylation and proteasomal degradation. The loss of TAOK3 had no effect on the abundance of SHP-2, which lacks a residue corresponding to SHP-1 threonine-394. Modulation of SHP-1 abundance by TAOK3 thus serves as a rheostat for TCR signaling and determines the activation threshold of T lymphocytes.

Targeting PknL as a novel therapeutic strategy against Mycobacterium tuberculosis

Abstract: INTRODUCTION: The bacterium “Mycobacterium tuberculosis (MTB)” causes tuberculosis, which is currently regarded as the most common cause of bacterial infection-related death globally. In addition to eukaryotes, MTB also encodes 11 “serine/threonine protein kinases (STPKs),” including PknA, B, C, D, E, F, G, H, J, K, and L that control cell division, cell development, metabolism, and dormancy. MATERIALS AND METHODS: The antisense and sense-orientated PknL have been cloned utilizing the shuttle mycobacterial vector-pAZI9018b, which is IPTG inducible. Utilizing the appropriate primers, the PknL (Rv2176) full-length gene from MTB has been amplified. The product which was amplified was then inoculated inside the vector at the “5’BamH1 and 3’Nde1” regions to produce PknL in the sense orientation (PknL-S) resembling PknL-S, and likewise, amplified substance was introduced into the vector at the 3’BamHI and 5’NdeI sites for producing PknL in the antisense orientation (PknL-AS). RESULTS: Regardless of the absence or presence of an inducer, PknL-AS had lower transcription levels than Wild type and PknL-S, but PknL-AS revealed a significant decrease in PknL expression during the end of 2nd week with 1 mM of IPTG and a further decrease on day 10 with 10 mM IPTG. In the presence of 10 mM IPTG, PknL expression has been close to, 1.8 log in Wild type, 1.4 log in PknL-AS, and 2.4 log in PknL-S. CONCLUSION: It has been clear that PknL may read environmental cues and respond appropriately, assisting bacteria to adapt to stressful situations in the host by lowering bacterial development, leading to a dormant state, and allowing bacteria to stay inside the host.

Lysophosphatidic acid down-regulates human RIPK4 mRNA in keratinocyte- derived cell lines.

The tight control of proliferating keratinocytes is vital to the successful function of the skin. Differentiation of dividing cells is necessary to form a skin barrier. The same dividing cells are necessary to heal wounds and when malignant form tumors. RIPK4, a serine-threonine kinase, plays critical roles in these processes. Its loss of function was associated with pathological keratinocyte proliferation and development of squamous cell carcinoma (SCC) in humans and mice. The current study extends previous findings in the importance of RIPK4 in keratinocyte proliferation. A serum-derived phospholipid, lysophosphatidic acid (LPA), was identified as an important biologic inhibitor of RIPK4. LPA functions by inhibiting the transcription of RIPK4 mRNA. LPA treatment led to increased keratinocyte proliferation, and this was compromised in cells with reduced RIPK4 expression. The current study may help to explain the mechanism by which RIPK4 was downregulated during SCC progression and provide insights on RIPK4 functions. It may also allow for targeting of RIPK4 through a natural component of serum.

Responses of transcriptome and metabolome in peanut leaves to dibutyl phthalate during whole growth period.

The application of agricultural film mulching technology has significantly contributed to increasing crop yield and income, but the pollution caused by residual film has seriously affected agricultural production and the natural environment. Agricultural film is commonly employed to enhance the yield of peanuts; its use may lead to excessive dibutyl phthalate (DBP) residues in peanut kernels. But, limited investigations have been conducted on the regulatory mechanism of peanut leaves in response to DBP exposure throughout the entire growth period. To bridge this knowledge gap, we investigated the differences in transcriptome and metabolome of peanut leaves under DBP stress. According to visual observations, the results of morphological response showed that the growth of peanut plants was significantly inhibited from seedling to pod stage under DBP treatment. Transcriptomic analysis results showed that the genes AH19G05510 (LRR receptor-like serine threonine-protein kinase) and AH20G31870 (disease resistance), belonging to the FAR1 family and bZIP family respectively, may be key genes involved in the resistance to DBP stress throughout its growth stages. Metabolomic analysis results showed that during the initial stage of DBP stress, the key metabolites in peanut leaves response to stress were carboxylic acids and derivatives, as well as fatty acyls. As peanut growth progressed, flavonoids gradually became more prominent in the resistance to DBP stress. By integrating metabolomics and transcriptomics analysis, we have identified that purine metabolism during seedling and flowering stages, as well as the flavone and flavonol biosynthesis pathways during pod and maturity stages, played a crucial role in response to DBP stress. These findings not only provide valuable key gene and metabolic information for studying anti-plasticizer pollution throughout the entire growth period of peanuts, but also offer reference for enhancing crop resistance to plasticizer pollution through genetic modification and metabolic regulation.

Discovery of a urea-based hit compound as a novel inhibitor of transforming growth factor-β type 1 receptor: in silico and in vitro studies.

Transforming growth factor β type 1 receptor (TGFβR1), a crucial serine-threonine kinase, is central to the TGFβ/Smad signaling pathway, governing cellular processes like growth, differentiation, apoptosis, and immune response. This pathway is closely linked to the epithelial-mesenchymal transition (EMT) process, which plays an important role in the metastasis of hepatocellular carcinoma (HCC). To date, only limited inhibitors targeting TGFβR1 have entered clinical trials, yet they encounter challenges, notably high toxicity, in clinical applications. Herein, an efficient virtual screening pipeline was developed. Eighty compounds were screened from a pool of over 17 million molecules based on docking scores and binding free energy. Four compounds were manually selected with the assistance of enhanced sampling method BPMD (binding pose metadynamics). The binding stability of these four compounds complexed with TGFβR1 was subsequently studied through long-timescale conventional molecular dynamics simulations. The three most promising compounds were subjected to in vitro bioactivity assays. Cpd272 demonstrated moderate inhibitory activity against TGFβR1, with an IC50 value of 1.57 ± 0.33 μM. Moreover, it exhibited cytotoxic effects on human hepatocellular carcinoma cell line Bel-7402. By shedding light on the binding mode of the receptor-ligand complexes, Cpd272 was identified as a hit compound featuring a novel urea-based scaffold capable of effectively inhibiting TGFβR1.

Chk2 Modulates Bmi1-Deficiency-Induced Renal Aging and Fibrosis via Oxidative Stress, DNA Damage, and p53/TGFβ1-Induced Epithelial-Mesenchymal Transition.

Renal aging may lead to fibrosis and dysfunction, yet underlying mechanisms remain unclear. We explored whether deficiency of the Polycomb protein Bmi1 causes renal aging via DNA damage response (DDR) activation, inducing renal tubular epithelial cell (RTEC) senescence and epithelial-mesenchymal transition (EMT). Bmi1 knockout mice exhibited oxidative stress, DDR activation, RTEC senescence, senescence-associated secretory phenotype (SASP), and age-related fibrosis in kidneys. Bmi1 deficiency impaired renal structure and function, increasing serum creatinine/urea, reducing creatinine clearance, and decreasing cortical thickness and glomerular number. However, knockout of the serine-threonine kinase Chk2 alleviated these aging phenotypes. Transcriptomics identified transforming growth factor beta 1 (TGFβ1) upregulation in Bmi1-deficient RTECs, but TGFβ1 was downregulated upon Chk2 knockout. The tumor suppressor protein p53 transcriptionally activated TGFβ1, promoting EMT in RTECs. Bmi1 knockout or oxidative stress (induced with H2O2) increased TGFβ1 expression, and EMT in RTECs and was partly reversed by p53 inhibition. Together, Bmi1 deficiency causes oxidative stress and DDR-mediated RTEC senescence/SASP, thus activating p53 and TGFβ1 to induce EMT and age-related fibrosis. However, blocking DDR (via Chk2 knockout) or p53 ameliorates these changes. Our study reveals mechanisms whereby Bmi1 preserves renal structure and function during aging by suppressing DDR and p53/TGFβ1-mediated EMT. These pathways represent potential targets for detecting and attenuating age-related renal decline.

Quercetin antagonized advanced glycated end products induced apoptosis and functional inhibition of fibroblasts from the prolapsed uterosacral ligament.

The altered behaviors and functions of pelvic floor fibroblasts are pathophysiological changes of pelvic organ prolapse (POP). Our previous study showed that advanced glycated end products (AGEs) accumulated in the pelvic tissues of POP and induced fibroblast apoptosis. The study was designed to investigate whether quercetin antagonize AGEs-induced apoptosis and functional inhibition of fibroblasts. The uptake of 5-ethynyl-2'-deoxyuridine (EdU) was evaluated for cell proliferation. Flow cytometric analysis was applied for cell apoptosis. Intracellular reactive oxygen species (ROS) content was determined by the fluorescence of dichlorofluorescein (DCF). The contractility of fibroblasts was measured by collagen gel contraction assay. The expressions of extracellular matrix (ECM) related genes and the expression of miR-4429 and caspase-3 were quantified by qPCR. The expressions of phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), serine-threonine kinase (Akt), and phosphorylated Akt (p-Akt) were analyzed by Western Blot. The down-regulation of miR-4429 was achieved by cell transfection. Quercetin antagonized AGEs-induced apoptosis, proliferation inhibition, and ROS increase in fibroblasts. Quercetin did not alleviate AGEs-induced contractile impairment of fibroblasts. Quercetin reduced the gene expressions of lysyl oxidase like protein 1 (LOXL1)and matrix metallopeptidase 1 (MMP1), and increased the gene expressions of lysyl oxidase (LOX) and fibrillin 2 (FBN2) in fibroblasts. Quercetin reversed AGEs-induced upregulation of PTEN and downregulation of PI3K, P-Akt, and miR-4429 in fibroblasts. The inhibitory effect of quercetin on AGEs-induced fibroblast apoptosis was inhibited by downregulating the expression of miR-4429. In conclusion, quercetin antagonized AGEs-induced apoptosis and functional inhibition of fibroblasts from the prolapsed uterosacral ligament. And inhibiting AGEs-induced down-regulation of miR-4429/PTEN/PI3K/Akt pathway was the mechanism underlying the antagonistic effect of quercetin on AGEs-induced apoptosis.

The Function of ASK1 in Sepsis and Stress-Induced Disorders.

Apoptosis signal-regulating kinase 1 (ASK1) is a serine-threonine kinase that is ubiquitously expressed in nucleated cells and is responsible for the activation of multiple mitogen-activated protein kinases (MAPK) to regulate cell stress. Activation of ASK1 via cellular stress leads to activation of downstream signaling components, activation of transcription factors, and proinflammatory cytokine production. ASK1 is also expressed in anucleate platelets and is a key player in platelet activation as it is important for signaling. Interestingly, the mechanism of ASK1 activation is cell type-dependent. In this review we will explore how ASK1 regulates a variety of cellular processes from innate immune function to thrombosis and hemostasis. We will discuss how ASK1 influences FcγRIIA-mediated platelet reactivity and how that reactivity drives platelet clearance. Furthermore, we will explore the role of ASK1 in thromboxane (TxA2) generation, which highlights differences in the way ASK1 functions in mouse and human platelets.

Brain-derived neurotrophic factor signaling in the neuromuscular junction during developmental axonal competition and synapse elimination.

During the development of the nervous system, there is an overproduction of neurons and synapses. Hebbian competition between neighboring nerve endings and synapses performing different activity levels leads to their elimination or strengthening. We have extensively studied the involvement of the brain-derived neurotrophic factor-Tropomyosin-related kinase B receptor neurotrophic retrograde pathway, at the neuromuscular junction, in the axonal development and synapse elimination process versus the synapse consolidation. The purpose of this review is to describe the neurotrophic influence on developmental synapse elimination, in relation to other molecular pathways that we and others have found to regulate this process. In particular, we summarize our published results based on transmitter release analysis and axonal counts to show the different involvement of the presynaptic acetylcholine muscarinic autoreceptors, coupled to downstream serine-threonine protein kinases A and C (PKA and PKC) and voltage-gated calcium channels, at different nerve endings in developmental competition. The dynamic changes that occur simultaneously in several nerve terminals and synapses converge across a postsynaptic site, influence each other, and require careful studies to individualize the mechanisms of specific endings. We describe an activity-dependent balance (related to the extent of transmitter release) between the presynaptic muscarinic subtypes and the neurotrophin-mediated TrkB/p75NTR pathways that can influence the timing and fate of the competitive interactions between the different axon terminals. The downstream displacement of the PKA/PKC activity ratio to lower values, both in competing nerve terminals and at postsynaptic sites, plays a relevant role in controlling the elimination of supernumerary synapses. Finally, calcium entry through L- and P/Q- subtypes of voltage-gated calcium channels (both channels are present, together with the N-type channel in developing nerve terminals) contributes to reduce transmitter release and promote withdrawal of the most unfavorable nerve terminals during elimination (the weakest in acetylcholine release and those that have already become silent). The main findings contribute to a better understanding of punishment-rewarding interactions between nerve endings during development. Identifying the molecular targets and signaling pathways that allow synapse consolidation or withdrawal of synapses in different situations is important for potential therapies in neurodegenerative diseases.

De novo variants underlying monogenic syndromes with intellectual disability in a neurodevelopmental cohort from India

The contribution of de novo variants as a cause of intellectual disability (ID) is well established in several cohorts reported from the developed world. However, the genetic landscape as well as the appropriate testing strategies for identification of de novo variants of these disorders remain largely unknown in low-and middle-income countries like India. In this study, we delineate the clinical and genotypic spectrum of 54 families (55 individuals) with syndromic ID harboring rare de novo variants. We also emphasize on the effectiveness of singleton exome sequencing as a valuable tool for diagnosing these disorders in resource limited settings. Overall, 46 distinct disorders were identified encompassing 46 genes with 51 single-nucleotide variants and/or indels and two copy-number variants. Pathogenic variants were identified in CREBBP, TSC2, KMT2D, MECP2, IDS, NIPBL, NSD1, RIT1, SOX10, BRWD3, FOXG1, BCL11A, KDM6B, KDM5C, SETD5, QRICH1, DCX, SMARCD1, ASXL1, ASXL3, AKT3, FBN2, TCF12, WASF1, BRAF, SMARCA4, SMARCA2, TUBG1, KMT2A, CTNNB1, DLG4, MEIS2, GATAD2B, FBXW7, ANKRD11, ARID1B, DYNC1H1, HIVEP2, NEXMIF, ZBTB18, SETD1B, DYRK1A, SRCAP, CASK, L1CAM, and KRAS. Twenty-four of these monogenic disorders have not been previously reported in the Indian population. Notably, 39 out of 53 (74%) disease-causing variants are novel. These variants were identified in the genes mainly encoding transcriptional and chromatin regulators, serine threonine kinases, lysosomal enzymes, molecular motors, synaptic proteins, neuronal migration machinery, adhesion molecules, structural proteins and signaling molecules.

Neuroprotective and Anti-inflammatory Effects of Rubiscolin-6 Analogs with Proline Surrogates in Position 2

Naturally occurring peptides, such as rubiscolins derived from spinach leaves, have been shown to possess some interesting activities. They exerted central effects, such as antinociception, memory consolidation and anxiolytic-like activity. The fact that rubiscolins are potent even when given orally makes them very promising drug candidates. The present work tested whether rubiscolin-6 (R-6, Tyr-Pro-Leu-Asp-Leu-Phe) analogs have neuroprotective and anti-inflammatory effects. These hypotheses were tested in the 6-hydroxydopamine (6-OHDA) injury model of human neuroblastoma SH-SY5Y and lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The determination of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), Caspase-3 activity, lipid peroxidation and nitric oxide (NO) production allowed us to determine the effects of peptides on hallmarks related to Parkinson’s Disease (PD) and inflammation. Additionally, we investigated the impact of R-6 analogs on serine-threonine kinase (also known as protein kinase B, AKT) and mammalian target of rapamycin (mTOR) activation. The treatment with analogs 3 (Tyr-Inp-Leu-Asp-Leu-Phe-OH), 5 (Dmt-Inp-Leu-Asp-Leu-Phe-OH) and 7 (Tyr-Inp-Leu-Asp-Leu-Phe-NH2) most effectively prevented neuronal death via attenuation of ROS, mitochondrial dysfunction and Caspase-3 activity. Peptides 5 and 7 significantly increased the protein expression of the phosphorylated-AKT (p-AKT) and phosphorylated-mTOR (p-mTOR). Additionally, selected analogs could also ameliorate LPS-mediated inflammation in macrophages via inhibition of intracellular generation of ROS and NO production. Our findings suggest that R-6 analogs exert protective effects, possibly related to an anti-oxidation mechanism in in vitro model of PD. The data shows that the most potent peptides can inhibit 6-OHDA injury by activating the PI3-K/AKT/mTOR pathway, thus playing a neuroprotective role and may provide a rational and robust approach in the design of new therapeutics or even functional foods.

Advances in the regulatory mechanisms of mTOR in necroptosis.

The mammalian target of rapamycin (mTOR), an evolutionarily highly conserved serine/threonine protein kinase, plays a prominent role in controlling gene expression, metabolism, and cell death. Programmed cell death (PCD) is indispensable for maintaining homeostasis by removing senescent, defective, or malignant cells. Necroptosis, a type of PCD, relies on the interplay between receptor-interacting serine-threonine kinases (RIPKs) and the membrane perforation by mixed lineage kinase domain-like protein (MLKL), which is distinguished from apoptosis. With the development of necroptosis-regulating mechanisms, the importance of mTOR in the complex network of intersecting signaling pathways that govern the process has become more evident. mTOR is directly responsible for the regulation of RIPKs. Autophagy is an indirect mechanism by which mTOR regulates the removal and interaction of RIPKs. Another necroptosis trigger is reactive oxygen species (ROS) produced by oxidative stress; mTOR regulates necroptosis by exploiting ROS. Considering the intricacy of the signal network, it is reasonable to assume that mTOR exerts a bifacial effect on necroptosis. However, additional research is necessary to elucidate the underlying mechanisms. In this review, we summarized the mechanisms underlying mTOR activation and necroptosis and highlighted the signaling pathway through which mTOR regulates necroptosis. The development of therapeutic targets for various diseases has been greatly advanced by the expanding knowledge of how mTOR regulates necroptosis.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis

Hyperplasia and migration of fibroblast-like synoviocytes (FLSs) are the key drivers in the pathogenesis of rheumatoid arthritis (RA) and joint destruction. Abundant Yes-associated protein (YAP), which is a powerful transcription co-activator for proliferative genes, was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms. Using Gene Expression Omnibus database analysis, it was found that Salvador homolog-1 (SAV1), the pivotal negative regulator of the Hippo-YAP pathway, was slightly downregulated in RA synovium. However, SAV1 protein expression is extremely reduced. Subsequently, it was revealed that SAV1 is phosphorylated, ubiquitinated, and degraded by interacting with an important serine-threonine kinase, G protein-coupled receptor (GPCR) kinase 2 (GRK2), which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E2 (PGE2) in RA. This process further contributes to the decreased phosphorylation, nuclear translocation, and transcriptional potency of YAP, and leads to aberrant FLSs proliferation. Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration. Similarly, paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations. Collectively, these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.