Phosphorylation Of Substrates Research Articles

The Switching of the Type of a ROS Signal from Mitochondria: The Role of Respiratory Substrates and Permeability Transition

Flashes of superoxide anion (O2−) in mitochondria are generated spontaneously or during the opening of the permeability transition pore (mPTP) and a sudden change in the metabolic state of a cell. Under certain conditions, O2− can leave the mitochondrial matrix and perform signaling functions beyond mitochondria. In this work, we studied the kinetics of the release of O2− and H2O2 from isolated mitochondria upon mPTP opening and the modulation of the metabolic state of mitochondria by the substrates of respiration and oxidative phosphorylation. It was found that mPTP opening leads to suppression of H2O2 emission and activation of the O2− burst. When the induction of mPTP was blocked by its antagonists (cyclosporine A, ruthenium red, EGTA), the level of substrates of respiration and oxidative phosphorylation and the selective inhibitors of complexes I and V determined the type of reactive oxygen species (ROS) emitted by mitochondria. It was concluded that upon complete and partial reduction and complete oxidation of redox centers of the respiratory chain, mitochondria emit H2O2, O2−, and nothing, respectively. The results indicate that the mPTP- and substrate-dependent switching of the type of ROS leaving mitochondria may be the basis for O2−- and H2O2-selective redox signaling in a cell.

Protein kinases as therapeutic targets for Alzheimer’s disease: a brief review

Alzheimer’s disease (AD) is a progressive and incurable neurodegenerative disorder, with an unknown etiology and a multifactorial pathophysiology characterized by protein misfolding, neuroinflammation, and neuronal loss. There are three well-discussed main hypotheses for the pathophysiology of AD, which are related to i) the accumulation of amyloid β (Aβ) protein aggregates in the extracellular space, ii) deposition of hyperphosphorylated tau fragments as neurofibrillary tangles, and iii) dysregulation of hemostasis of some neurotransmitters involved in the disease, such as acetylcholine (ACh) and glutamate. The association of all these factors is responsible for installing oxidative stress and neuroinflammation, which contribute to progressive neuronal death in specific brain regions. More recently, other remarkable pathological characteristics have been described, involving changes in all levels of cellular components, especially in the action and function of protein kinases. These enzymes are crucial for cellular regulation since they play a pivotal role in the phosphorylation of protein substrates by transferring a phosphate group from the ATP molecule to threonine, serine, or tyrosine residues. In more recent studies, some kinases have been especially reported by their role in inflammatory and oxidative processes associated to AD, such as cAMP-dependent protein kinase A (PKA), cyclin-dependent protein kinase 5 (CDK5), glycogen synthase kinase 3β (GSK-3β), and the microtubule affinity regulatory kinases (MARKs). Under homeostatic conditions, protein kinases act as cellular signals, directing physiological responses, but in AD pathogenesis, these enzymes have an exacerbated activity in the brain, justifying the need for a better comprehension of their function and role, and how new kinase inhibitors could lead to innovative drugs. In this context, this brief review aimed to compile the literature data related to the most recent efforts and strategies in Medicinal Chemistry in the discovery of new kinase inhibitors, opening new ways to AD therapeutics.

KRAS-mediated upregulation of CIP2A promotes suppression of PP2A-B56α to initiate pancreatic cancer development.

Oncogenic mutations in KRAS are present in ~95% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) and are considered the initiating event of pancreatic intraepithelial neoplasia (PanIN) precursor lesions. While it is well established that KRAS mutations drive the activation of oncogenic kinase cascades during pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (PP2A) has been implicated in suppressing KRAS-driven cellular transformation and low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRASG12D induces the expression of an endogenous inhibitor of PP2A activity, Cancerous Inhibitor of PP2A (CIP2A), and phosphorylation of the PP2A substrate, c-MYC. Consistent with these findings, KRASG12D sequestered the specific PP2A subunit responsible for c-MYC degradation, B56α, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation in vivo, knockout of B56α promoted KRASG12D tumorigenesis by accelerating acinar-to-ductal metaplasia (ADM) and the formation of PanIN lesions. The process of ADM was attenuated ex vivo in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (SMAPs). Together, our results suggest that suppression of PP2A-B56α through KRAS signaling can promote the MYC-driven initiation of pancreatic tumorigenesis.

Light induces a rapid increase in cAMP and activates PKA in rod outer segments of the frog retina.

The phototransduction cascade enables the photoreceptor to detect light over a wide range of intensities without saturation. The main second messenger of the cascade is cGMP and the primary regulatory mechanism is calcium feedback. However, some experimental data suggest that cAMP may also play a role in regulating the phototransduction cascade, but this would require changes in cAMP on a time scale of seconds. Currently, there is a lack of data on the dynamics of changes in intracellular cAMP levels on this timescale. This is largely due to the specificity of the sensory modality of photoreceptors, which makes it practically impossible to use conventional experimental approaches based on fluorescence methods. In this study, we employed the method of rapid cryofixation of retinal samples after light stimulation and subsequent isolation of outer segment preparations. The study employed highly sensitive metabolomics approaches to measure levels of cAMP. Additionally, PKA activity was measured in the samples using a western blot. The results indicate that when exposed to near-saturating but still moderate light, cAMP levels increase transiently within the first second and then return to pre-stimulus levels. The increase in cAMP activates PKA, resulting in the phosphorylation of PKA-specific substrates in frog retinal outer segments.

The LAT Rheostat as a Regulator of Megakaryocyte Activation.

Specifically positioned negatively charged residues within the cytoplasmic domain of the adaptor protein, linker for the activation of T cells (LAT), have been shown to be important for efficient phosphorylation of tyrosine residues that function to recruit cytosolic proteins downstream of immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling. LAT tyrosine 132-the binding site for PLC-γ2-is a notable exception, preceded instead by a glycine, making it a relatively poor substrate for phosphorylation. Mutating Gly131 to an acidic residue has been shown in T cells to enhance ITAM-linked receptor-mediated signaling. Whether this is generally true in other cell types is not known. To examine whether LAT Gly131 restricts ITAM signaling in cells of the megakaryocyte lineage, we introduced an aspartic acid at this position in human induced pluripotent stem cells (iPSCs), differentiated them into megakaryocytes, and examined its functional consequences. iPSCs expressing G131D LAT differentiated and matured into megakaryocytes normally, but exhibited markedly enhanced reactivity to glycoprotein VI (GPVI)-agonist stimulation. The rate and extent of LAT Tyr132 and PLC-γ2 phosphorylation, and proplatelet formation on GPVI-reactive substrates, were also enhanced. These data demonstrate that a glycine residue at the -1 position of LAT Tyr132 functions as a kinetic bottleneck to restrain Tyr132 phosphorylation and signaling downstream of ITAM receptor engagement in the megakaryocyte lineage. These findings may have translational applications in the burgeoning field of in vitro platelet bioengineering.

Ligand-induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity.

γ-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA). Like smaller-type analogs, PIPA binding to the CaMKIIα hub domain promoted thermal stability. PIPA additionally modulated CaMKIIα activity under sub-maximal CaM concentrations and ultimately led to reduced substrate phosphorylation. A high-resolution X-ray crystal structure of a stabilized CaMKIIα (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small-angle X-ray scattering (SAXS) solution structures and mass photometry of the CaMKIIα wild-type hub domain in the presence of PIPA revealed a high degree of ordered self-association (stacks of CaMKIIα hub domains). This stacking neither occurred with the smaller compound 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKIIα W403L hub was stabilized to a larger extent by PIPA compared to CaMKIIα hub wild type, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand-induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKIIα kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKIIα activity via the hub domain.

GpsB Coordinates StkP Signaling as a PASTA Kinase Adaptor in Streptococcus pneumoniae Cell Division

StkP, the Ser/Thr protein kinase of the major human pathogen Streptococcus pneumoniae, monitors cell wall signals and regulates growth and division in response. In vivo, StkP interacts with GpsB, a cell division protein required for septal ring formation and closure, that affects StkP-dependent phosphorylation. Here, we report that although StkP has basal intrinsic kinase activity, GpsB promotes efficient autophosphorylation of StkP and phosphorylation of StkP substrates. Phosphoproteomic analyzes showed that GpsB is phosphorylated at several Ser and Thr residues. We confirmed that StkP directly phosphorylates GpsB in vitro and in vivo, with T79 and T83 being the major phosphorylation sites. In vitro, phosphoablative GpsB substitutions had a lower potential to stimulate StkP activity, whereas phosphomimetic substitutions were functional in terms of StkP activation. In vivo, substitutions of GpsB phosphoacceptor residues, either phosphoablative or mimetic, had a negative effect on GpsB function, resulting in reduced StkP-dependent phosphorylation and impaired cell division. The bacterial two-hybrid assay and co-immunoprecipitation of GpsB from cells with differentially active StkP indicated that increased phosphorylation of GpsB resulted in a more efficient interaction of GpsB with StkP. Our data suggest that GpsB acts as an adaptor that directly promotes StkP activity by mediating interactions within the StkP signaling hub, ensuring StkP recruitment into the complex and substrate specificity. We present a model that interaction of StkP with GpsB and its phosphorylation and dephosphorylation dynamically modulate kinase activity during exponential growth and under cell wall stress of S. pneumoniae, ensuring the proper functioning of the StkP signaling pathway.

Combined inhibition of CDK4/6 and AKT is highly effective against the luminal androgen receptor (LAR) subtype of triple negative breast cancer

Luminal Androgen Receptor (LAR) triple-negative breast cancers (TNBC) express androgen receptors (AR), exhibit high frequency of PIK3CA mutations and intact RB. Herein, we investigated combined blockade of the CDK4/6 and PI3K signaling with palbociclib, alpelisib, and capivasertib, which inhibit CDK4/6, PI3Kα, and AKT1-3, respectively. The combination of palbociclib/capivasertib, but not palbociclib/alpelisib, synergistically inhibited proliferation of MDA-MB-453 and MFM-223 LAR cells [synergy score 7.34 (p = 5.81x10−11) and 4.78 (p = 0.012), respectively]. The AR antagonist enzalutamide was inactive against MDA-MB-453, MFM-223, and CAL148 cells and did not enhance the efficacy of either combination. Palbociclib/capivasertib inhibited growth of LAR patient-derived xenografts more potently than palbociclib/alpelisib. Treatment of LAR cells with palbociclib suppressed phosphorylated-RB and resulted in adaptive phosphorylation/activation of S473 pAKT and AKT substrates GSK3β, PRAS40, and FoxO3a. Capivasertib blocked palbociclib-induced phosphorylation of AKT substrates more potently than alpelisib. Treatment with PI3Kβ inhibitors did not block phosphorylation of AKT substrates, suggesting that PI3Kβ did not mediate the adaptive response to CDK4/6 inhibition. Phosphokinase arrays of MDA-MB-453 cells treated with palbociclib showed time-dependent upregulation of PDGFRβ, GSK3β, STAT3, and STAT6. RNA silencing of PDGFRβ in palbociclib-treated MDA-MB-453 and MFM-223 cells blocked the upregulation of S473 pAKT, suggesting that the adaptive response to CDK4/6 blockade involves PDGFRβ signaling. Finally, treatment with palbociclib and the PDGFR inhibitor CP637451 arrested growth of MDA-MB-453 and MFM-223 cells to the same degree as palbociclib/capivasertib. These findings support testing the combination of CDK4/6 and AKT inhibitors in patients with LAR TNBC, and further investigation of PDGFR antagonists in this breast cancer subtype.

Targeting Specific Kinase Substrates Rescues Increased Colitis Severity Induced by the Crohn's Disease-Linked LRRK2-N2081D Variant.

LRRK2 contains a kinase domain where both the N2081D Crohn's disease (CD) risk and the G2019S Parkinson's disease (PD)-pathogenic variants are located. The mechanisms by which the N2081D variant increase CD risk, and how these adjacent mutations result in distinct diseases, remain unclear. To investigate the pathophysiology of the CD-linked LRRK2 N2081D variant, we generated a knock-in (KI) mouse model and compared its effects to those of the LRRK2-G2019S mutation. We find that Lrrk2 N 2081 D KI mice demonstrate heightened sensitivity to induced colitis, resulting in more severe inflammation and intestinal damage than Lrrk2 G2019S KI and wild-type mice. Analysis of Colon tissue revealed distinct mutation-dependent LRRK2 RAB substrate phosphorylation, with significantly elevated phosphorylated RAB10 levels in Lrrk2 N2081D mice. In cells, we demonstrate that the N2081D mutation activates LRRK2 through a mechanism distinct from that of LRRK2-G2019S. We further find that proinflammatory stimulation enhances LRRK2 kinase activity, leading to mutation-dependent differences in RAB phosphorylation and inflammatory responses in dendritic cells. Finally, we show that genetic knockout of Rab12 , but not pharmacological LRRK2 kinase inhibition, significantly reduced colitis severity in Lrrk2 N2081D mice. Our study characterizes the pathogenic mechanisms of LRRK2-linked CD, highlights important structural and functional differences between disease-associated LRRK2 variants, and suggests RAB proteins as promising therapeutic targets for modulating LRRK2 activity in CD treatment.

The Value IRS-1 rs1801278G > A Polymorphism Testing in Evaluating Infertile Women with Polycystic Ovarian Syndrome: A Case-control Study

Background: Polycystic ovary syndrome (PCOS) is a leading cause of infertility. Insulin resistance is a key element in pathogenesis. The insulin receptor causes phosphorylation of the insulin receptor substrate (IRS); IRS-1 rs1801278G > A polymorphism variant is the most common genetic variant associated with IR and PCOS. Objective: We aimed to examine the frequency of IRS-1 rs1801278G > A polymorphism variant and test its value in evaluating infertile PCOS women. Methods: A case-control study recruited 140 age and body-mass-matched participants in the university hospital, subdivided according to Rotterdam criteria into PCOS cases (70/140) and healthy controls (70/140). We collected demographic data, ultrasonic [antral follicles and endometrial thickness], hormonal [FSH, LH, AMH, E2], and genetic data by polymerase chain reaction for analysis. Result: Wild GG SNP rs1801278 G was meaningfully higher among controls (58.57%, P<0.0001). Mutant AA SNP rs1801278 was significantly higher in PCOS women (37.14%, P-value =0.0001, an odds ratio of 20.50, 95% CI (9.42-28.63) to develop PCOS. Heterogenous GA gene SNP rs1801278 showed a trend of higher frequency in PCOS patients with 44.29%; OR of 3.91, 95% CI (1.37–7.55); P = 0.422. Upon correlating infertility parameters to SNP rs1801278 G>A polymorphism, statistical differences were found with AFC, LH/FSH ratio, and serum testosterone. As for the AMH, E2, and endometrial thickness, they failed to have a statistical value. Conclusion: The significant correlation of genetic polymorphism to infertility parameters among PCOS women opens a new therapeutic and prognostic avenue that helps gynecologists tailor manganate for a better and safer outcome.

Targeted Degradation of Protein Kinase A via a Stapled Peptide PROTAC.

Proteolysis-targeting chimeras (PROTACs) are bifunctional molecules that bind and recruit an E3 ubiquitin ligase to a targeted protein of interest, often through the utilization of a small molecule inhibitor. To expand the possible range of kinase targets that can be degraded by PROTACs, we sought to develop a PROTAC utilizing a hydrocarbon-stapled peptide as the targeting agent to bind the surface of a target protein of interest. In this study, we describe the development of a proteolysis-targeting chimera, dubbed Stapled Inhibitor Peptide - PROTAC or StIP-TAC, linking a hydrocarbon-stapled peptide with an E3 ligase ligand for targeted degradation of Protein Kinase A (PKA). This StIP-TAC molecule stimulated E3-mediated protein degradation of PKA, and this effect could be reversed by the addition of the proteasomal inhibitor MG-132. Further, StIP-TAC treatment led to a significant reduction in PKA substrate phosphorylation. Since many protein targets of interest lack structural features that make them amenable to small molecule targeting, development of StIP-TACs may broaden the potential range of protein targets using a PROTAC-mediated proteasomal degradation approach.

16 Linking VHL and SETD2 in a common oncogenic pathway that converges on the mitoticspindle

Abstract Background Loss of chromosome 3p is a landmark event in clear cell renal cell carcinoma (ccRCC) that results in mono-allelic loss of VHL (von Hippel Lindau) and SETD2 (Set-domain containing 2) (and other tumor suppressors co-located on 3p). Second hits in VHL inactivate this key tumor suppressor initiating tumor progression. SETD2, a histone methyltransferase, was previously shown to have a dual function in methylating both histones and microtubules, thereby contributing to both the histone and tubulin codes. Methylation by SETD2 on microtubules occurs at the mitotic spindle and is essential for normal mitosis and cytokinesis, with loss of SETD2 acting as a strong driver of apoptosis. This raises a conundrum of how cancer cells survive early mono-allelic loss of SETD2, escaping cell death. Methods Using biochemical kinase assays and mass spectrometry we have identified SETD2 as a substrate for AURKA. In addition, we have used immunoblotting and immunofluorescence assays to probe phosphorylation on SETD2 and the impact of phosphorylation of SETD2 both on its chromatin and cytoskeleton targets. Results We have identified the mitotic kinase, Aurora kinase A (AURKA), as a regulator of SETD2. Our data uncover SETD2 as a unique substrate for phosphorylation by AURKA, with mass spectrometry identifying serine 2080 (S2080) as the site of phosphorylation on SETD2. We found phosphorylation of SETD2 by AURKA at S2080 contributes to its methyltransferase (i.e. enzymatic) activity on microtubules but does not impact chromatin methylation on H3K36 which remains unaltered. We show that VHL regulates SETD2 via AURKA, and loss of phosphorylation on SETD2 results in mitotic defects and genomic instability. Importantly, we demonstrate that inhibition of AURKA is synthetic lethal in the setting of VHL and SETD2 deficiency. Conclusions AURKA expression levels are high in VHL-null cells resulting from an inability of VHL to target AURKA for degradation and our data now highlight a direct link between VHL and SETD2, two tumor suppressors believed to independently drive RCC pathogenesis. In summary, our data reveal a tumor-specific vulnerability linked to mitotic fragility that can be precisely targeted to ultimately drive mitotic catastrophe. DOD CDMRP Funding: yes

Rutin alleviates advanced glycosylation end products-induced insulin resistance by inhibiting SOCS3/IRS1 and activating PI3K/AKT signaling pathways in HepG2 cells

The accumulation of advanced glycosylation end products (AGEs) from food in the body disrupts normal physiological insulin activity and causes insulin resistance. This study compared the effects of ten types of polyphenols on AGEs production in both chemical and food models. It also investigated the impact of polyphenols on AGEs-induced insulin resistance in HepG2 cell models. Results showed that luteolin had the strongest inhibitory effect on fluorescent AGEs in the three chemical models. Rutin exhibited the most potent capacity to inhibit AGEs in biscuit models. Moreover, rutin alleviated the glucose metabolism disorders induced by representative AGEs (methylglyoxal-modified bovine serum albumin, MGO-BSA) in HepG2 cells. Mechanistic studies revealed that rutin could inhibit the phosphorylation of insulin receptor substrate 1 (IRS1) and the activity of suppressor of cytokine signaling 3 (SOCS3), while activating the phosphorylation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) to alleviate the MGO-BSA-induced insulin resistance in HepG2 cells.

TPCA-1 compound, inhibiting testis-specific serine/threonine protein kinase 3 for potential male sterile in Bombyx mori.

Protein kinases are a type of transferase enzyme that catalyze the phosphorylation of protein substrates, including receptor proteins. Testis-specific serine/threonine kinases (TSSKs) are a highly conserved group of protein kinases found in various organisms. They play an essential role in male reproduction by influencing sperm development and function. In this study, we report on the characterization of BmTSSK3, a TSSK from the silkworm, Bombyx mori. We found that BmTSSK3 is specifically expressed in the testis and localized to the sperm flagella, particularly in the sperm tail cyst. Furthermore, we developed BmTSSK3 inhibitors through molecular docking and binding assays. Small molecules 5-(4-Fluorophenyl)-2-ureidothiophene-3-carboxamide (TPCA-1)and Imidurea were identified to bind to BmTSSK3. Using site-specific mutation technology, we identified amino acid residues R134 and S184 as crucial binding sites for small molecules. RNA interference assay and Western blot analysis showed that knockdown of BmTSSK3 significantly decreased histone 3 phosphorylation. To confirm the inhibitory effect of these small molecules, we treated silkworm testes with TPCA-1 and observed a strong inhibitory effect. TPCA-1 is an inhibitor of BmTSSK3, which raises its potential as a future candidate for male sterility of the silkworm. Thus, this study may offer a novel strategy for sterile silkworms as well as insects. © 2024 Society of Chemical Industry.

Coordinated Regulation of Renal Glucose Reabsorption and Gluconeogenesis by mTORC2 and Potassium.

The kidney proximal tubule is uniquely responsible for reabsorption of filtered glucose and gluconeogenesis (GNG). Insulin stimulates glucose transport and suppresses GNG in the proximal tubule, however, the signaling mechanisms and coordinated regulation of these processes remain poorly understood. The kinase complex mTORC2 is critical for regulation of growth, metabolism, solute transport, and electrolyte homeostasis in response to a wide array of inputs. Here we examined its role in the regulation of renal glucose reabsorption and GNG. Rictor, an essential component of mTORC2, was knocked out using the Pax8-LC1 system to generate inducible tubule specific Rictor knockout (TRKO) mice. These animals were subjected to fasting, refeeding, and variation in dietary K + . Metabolic parameters including glucose homeostasis and renal function were assessed in balance cages. Kidneys and livers were also harvested for molecular analysis of gluconeogenic enzymes, mTORC2-regulated targets, and plasma membrane glucose transporters. On a normal chow diet, TRKO mice had marked glycosuria despite indistinguishable blood glucose relative to WT controls. Kidney plasma membrane showed lower SGLT2 and SGLT1 in the fed state, supporting reduced renal glucose reabsorption. Additional metabolic testing provided evidence for renal insulin resistance with elevated fasting insulin, impaired pyruvate tolerance, elevated hemoglobin A1c, and increased renal gluconeogenic enzymes in the fasted and fed states. These effects were correlated with reduced downstream phosphorylation of Akt and the transcription factor FOXO4, identifying a novel role of FOXO4 in the kidney. Interestingly, high dietary K + prevented glycosuria and excessive GNG in TRKO mice, despite persistent reduction in mTORC2 substrate phosphorylation. Renal tubule mTORC2 is critical for coordinated regulation of sodium-glucose cotransport by SGLT2 and SGLT1 as well as renal GNG. Dietary K + promotes glucose reabsorption and suppresses GNG independently of insulin signaling and mTORC2, potentially providing an alternative signaling mechanism in states of insulin resistance. The kidney contributes to regulation of blood glucose through reabsorption of filtered glucose and gluconeogenesis. This study shows that mTORC2 and dietary potassium coordinate the regulation of sodium-glucose cotransport and glucose production in the kidney via independent mechanisms. New insights into the regulation of these processes in the kidney offer promising implications for diabetes mellitus management and treatment.

Exploring drug repositioning possibilities of kinase inhibitors via molecular simulation.

Kinases, a class of enzymes controlling various substrates phosphorylation, are pivotal in both physiological and pathological processes. Although their conserved ATP binding pockets pose challenges for achieving selectivity, this feature offers opportunities for drug repositioning of kinase inhibitors (KIs). This study presents a cost-effective in silico prediction of KIs drug repositioning via analyzing cross-docking results. We established the KIs database (278 unique KIs, 1834 bioactivity data points) and kinases database (357 kinase structures categorized by the DFG motif) for carrying out cross-docking. Comparative analysis of the docking scores and reported experimental bioactivity revealed that the Atypical, TK, and TKL superfamilies are suitable for drug repositioning. Among these kinase superfamilies, Olverematinib, Lapatinib, and Abemaciclib displayed enzymatic activity in our focused AKT-PI3K-mTOR pathway with IC50 values of 3.3, 3.2 and 5.8 μM. Further cell assays showed IC50 values of 0.2, 1.2 and 0.6 μM in tumor cells. The consistent result between prediction and validation demonstrated that repositioning KIs via in silico method is feasible.

Clinical characteristics and pathophysiological properties of newly discovered LRRK2 variants associated with Parkinson's disease

Leucine-rich repeat kinase 2 (LRRK2) is the most common gene responsible for familial Parkinson's disease (PD). The gene product of LRRK2 contains multiple protein domains, including armadillo repeat, ankyrin repeat, leucine-rich repeat (LRR), Ras-of-complex (ROC), C-terminal of ROC (COR), kinase, and WD40 domains. In this study, we performed genetic screening of LRRK2 in our PD cohort, detecting sixteen LRRK2 rare variants. Among them, we selected seven variants that are likely to be familial and characterized them in terms of LRRK2 protein function, along with clinical information and one pathological analysis. The seven variants were S1120P and N1221K in the LRR domain; I1339M, S1403R, and V1447M in the ROC domain; and I1658F and D1873H in the COR domain. The kinase activity of the LRRK2 variants N1221K, S1403R, V1447M, and I1658F toward Rab10, a well-known phosphorylation substrate, was higher than that of wild-type LRRK2. LRRK2 D1873H showed enhanced self-association activity, whereas LRRK2 S1403R and D1873H showed reduced microtubule-binding activity. Pathological analysis of a patient with the LRRK2 V1447M variant was also performed, which revealed Lewy pathology in the brainstem. No functional alterations in terms of kinase activity, self-association activity, and microtubule-binding activity were detected in LRRK2 S1120P and I1339M variants. However, the patient with PD carrying LRRK2 S1120P variant also had a heterozygous Glucosylceramidase beta 1 (GBA1) L444P variant. In conclusion, we characterized seven LRRK2 variants potentially associated with PD. Five of the seven variants in different LRRK2 domains exhibited altered properties in kinase activity, self-association, and microtubule-binding activity, suggesting that each domain variant may contribute to disease progression in different ways.

Impaired Sonic Hedgehog Responsiveness of Induced Pluripotent Stem Cell-Derived Floor Plate Cells Carrying the LRRK2-I1371V Mutation Contributes to the Ontogenic Origin of Lower Dopaminergic Neuron Yield.

Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson's disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.

Effect of PLCE1-driven aerobic glycolysis through ENO1 modulation on immune evasion in esophageal squamous cell carcinoma (ESCC).

e16016 Background: Our prior investigations have demonstrated that PLCE1 actively promotes tumorigenesis, angiogenesis, and cell cycle progression in ESCC. However, the precise role of PLCE1 in glycolysis and its influence on the tumor immune microenvironment (TIME) remain elusive. Methods: To elucidate potential pathways regulated by PLCE1, we applied Affymetrix GeneChip, Affymetrix ClariomD Array, and IP-MS techniques to identify molecules interacting with PLCE1. Subsequently, we validated the involement of PLCE1 in glycolysis through assessments of glycolytic product production in both in vivo and in vitro assays. The underlying mechanisms by which PLCE1 influences glycolysis were further probed through a combination of IP assays, tumor growth experiments, and ubiquitination assays. Next, we conducted multi-color immunofluorescence staining to examine the infiltration of immune cells in human ESCC tissues. We utilized a transgenic PLCE1-/- C57BL/6 mice model to investigate the immune cell distribution in TIME. Results: PLCE1 emerges as a pivotal regulator in glycolytic processes within ESCC cell lines. Knockdown of PLCE1 instigates a notable attenuation of glycolysis, achieved through the modulation of ENO1 expression, a key enzyme in glycolysis. This effect was also observed in vivo. We have elucidated a novel pathway in which PLCE1 orchestrates a reduction in the expression of FBXW7, an E3 ubiquitin ligase, thereby attenuating FBXW7-mediated ubiquitination of ENO1 and fostering ENO1 stability. Based on mutations identified through sequencing of ESCC tissue and drawing from previous studies, we have pinpointed the specific interacting sites between FBXW7 and ENO1. Substrate phosphorylation is necessary for the recognition of FBXW7. We unveil a mechanism wherein PLCE1 interacts with CDK2 to augment ENO1 phosphorylation, potentiating FBXW7 recognition. Nonetheless, CDK2-mediated ENO1 phosphorylation fails to elicit FBXW7-mediated ubiquitination due to limited FBXW7 expression in ESCC. Elevated tumor glycolysis induced by PLCE1 via ENO1 engenders heightened lactate production, thereby dampening cytokine production and CD8+ T cell activation. In human ESCC tissues, a diminished CD8+ T cell population is observed proximal to PLCE1+ENO1+ tumor cells, concomitant with an augmented accumulation of exhausted CD8+ PD1+ T cells in their vicinity. In an ESCC murine model, a more robust infiltration and cytokine production, coupled with diminished PD-1 expression on both CD4+ and CD8+ T cells, is noted in the PLCE1-/- genotype. This effect was particularly prominent upon ENO1 inhibitor administration. Conclusions: Our investigation elucidates that PLCE1 orchestrates the glycolytic pathway in ESCC by upregulating the expression of ENO1, consequently hindering the infiltration and activation of CD8+ T cells, thus fostering an immunosuppressive TIME.

Ketogenic diet administration later in life improves memory by modifying the synaptic cortical proteome via the PKA signaling pathway in aging mice

Aging compromises brain function leading to cognitive decline. A cyclic ketogenic diet (KD) improves memory in aged mice after long-term administration; however, short-term effects later in life and the molecular mechanisms that govern such changes remain unclear. Here, we explore the impact of a short-term KD treatment starting at elderly stage on brain function of aged mice. Behavioral testing and long-term potentiation (LTP) recordings reveal that KD improves working memory and hippocampal LTP. Furthermore, the synaptosome proteome of aged mice fed a KD long-term evidence changes predominantly at the presynaptic compartment associated to the protein kinase A (PKA) signaling pathway. These findings were corroborated invivo by western blot analysis, with high BDNF abundance and PKA substrate phosphorylation. Overall, we show that a KD modifies brain function even when it is administered later in life and recapitulates molecular features of long-term administration, including the PKA signaling pathway, thus promoting synaptic plasticity at advanced age.