Background
Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disorder of the exocrine pancreatic gland. A previous study from this laboratory showed that ethanol (EtOH) causes ...cytotoxicity, dysregulates AMPKα and ER/oxidative stress signaling, and induces inflammatory responses in primary human pancreatic acinar cells (hPACs). Here we examined the differential cytotoxicity of EtOH and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters; FAEEs) metabolites in hPACs was examined to understand the metabolic basis and mechanism of ACP.
Methods
We evaluated concentration‐dependent cytotoxicity, AMPKα inactivation, ER/oxidative stress, and inflammatory responses in hPACs by incubating them for 6 h with EtOH, acetaldehyde, or FAEEs at clinically relevant concentrations reported in alcoholic subjects using conventional methods. Cellular bioenergetics (mitochondrial stress and a real‐time ATP production rate) were determined using Seahorse XFp Extracellular Flux Analyzer in AR42J cells treated with acetaldehyde or FAEEs.
Results
We observed concentration‐dependent increases in LDH release, inactivation of AMPKα along with upregulation of ACC1 and FAS (key lipogenic proteins), downregulation of p‐LKB1 (an oxidative stress‐sensitive upstream kinase regulating AMPKα) and CPT1A (involved in β‐oxidation of fatty acids) in hPACs treated with EtOH, acetaldehyde, or FAEEs. Concentration‐dependent increases in oxidative stress and ER stress as measured by GRP78, unspliced XBP1, p‐eIF2α, and CHOP along with activation of p‐JNK1/2, p‐ERK1/2, and p‐P38MAPK were present in cells treated with EtOH, acetaldehyde, or FAEEs, respectively. Furthermore, a significant decrease was observed in the total ATP production rate with subsequent mitochondrial stress in AR42J cells treated with acetaldehyde and FAEEs.
Conclusions
EtOH and its metabolites, acetaldehyde and FAEEs, caused cytotoxicity, ER/oxidative and mitochondrial stress, and dysregulated AMPKα signaling, suggesting a key role of EtOH metabolism in the etiopathogenesis of ACP. Because oxidative EtOH metabolism is negligible in the exocrine pancreas, the pathogenesis of ACP could be attributable to the formation of FAEEs and related pancreatic acinar cell injury.
To better understand the etiology of alcoholic chronic pancreatitis (ACP), we investigated the cytotoxicity of ethanol and its metabolites in primary human pancreatic acinar cells (hPACs). Ethanol, as well as its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters; FAEEs) metabolites, caused injury, endoplasmic reticulum/oxidative stress and dysregulated AMPKα signaling in hPACs. In view of negligible oxidative and substantial nonoxidative metabolism of ethanol in the pancreatic gland during chronic alcohol ingestion, FAEEs could significantly contribute to the etiopathogenesis of ACP.
We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations ...(Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA.
Owing to the dysregulation of protein kinase activity in many diseases including cancer, this enzyme family has become one of the most important drug targets in the 21st century. There are 68 ...FDA-approved therapeutic agents that target about two dozen different protein kinases and six of these drugs were approved in 2021. Of the approved drugs, twelve target protein-serine/threonine protein kinases, four are directed against dual specificity protein kinases (MEK1/2), thirteen block nonreceptor protein-tyrosine kinases, and 39 target receptor protein-tyrosine kinases. The data indicate that 58 of these drugs are prescribed for the treatment of neoplasms (49 against solid tumors including breast, lung, and colon, five against nonsolid tumors such as leukemias, and four against both solid and nonsolid tumors: acalabrutinib, ibrutinib, imatinib, and midostaurin). Three drugs (baricitinib, tofacitinib, upadacitinib) are used for the treatment of inflammatory diseases including rheumatoid arthritis. Of the 68 approved drugs, eighteen are used in the treatment of multiple diseases. The following six drugs received FDA approval in 2021 for the treatment of these specified diseases: belumosudil (graft vs. host disease), infigratinib (cholangiocarcinomas), mobocertinib and tepotinib (specific forms of non-small cell lung cancer), tivozanib (renal cell carcinoma), and trilaciclib (to decrease chemotherapy-induced myelosuppression). All of the FDA-approved drugs are orally effective with the exception of netarsudil, temsirolimus, and the newly approved trilaciclib. This review summarizes the physicochemical properties of all 68 FDA-approved small molecule protein kinase inhibitors including lipophilic efficiency and ligand efficiency.
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Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report ...unique Ca
signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca
-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and proliferation. Conditional cpk10 cpk30 cpk32 and nlp7 mutants similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca
signalling network integrates transcriptome and cellular metabolism with shoot-root coordination and developmental plasticity in shaping organ biomass and architecture.
This study examines the role of protein kinase C (PKC) and AMP‐activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad‐spectrum PKC ...inhibitors (Ro‐31‐8245, Go6983), protected against APAP cytotoxicity despite sustained c‐jun‐N‐terminal kinase (JNK) activation. Broad‐spectrum PKC inhibitor treatment enhanced p‐AMPK levels and AMPK regulated survival‐energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p‐AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro‐31‐8245 treatment in mice up‐regulated p‐AMPK levels, increased autophagy (i.e., increased LC3‐II formation, p62 degradation), and protected against APAP‐induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC‐α using antisense (ASO) in mice also protected against APAP‐induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC‐α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP‐induced PKC‐α translocation to mitochondria, suggesting PKC‐α and JNK interplay in a feed‐forward mechanism to mediate APAP‐induced liver injury. Conclusion: PKC‐α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP‐induced liver injury. (Hepatology 2014;59:1543‐1554)
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Because mutations, overexpression, and dysregulation of protein kinases play essential roles in the pathogenesis of many illnesses, this enzyme family has become one of the most ...important drug targets in the past 20 years. The US FDA has approved 48 small molecule protein kinase inhibitors, nearly all of which are orally effective with the exceptions of netarsudil (which is given as an eye drop) and temsirolimus (which is given intravenously). Of the 48 approved drugs, the majority (25) target receptor protein-tyrosine kinases, ten target non-receptor protein-tyrosine kinases, and 13 target protein-serine/threonine protein kinases. The data indicate that 43 of these drugs are used in the treatment of malignancies (36 against solid tumors including lymphomas and seven against non-solid tumors, e.g., leukemias). Seven drugs are used in the treatment of non-malignancies: baricitinib, rheumatoid arthritis; fostamatinib, chronic immune thrombocytopenia; ruxolitinib, myelofibrosis and polycythemia vera; nintedanib, idiopathic pulmonary fibrosis; sirolimus, renal graft vs. host disease; netarsudil, glaucoma; tofacitinib, rheumatoid arthritis, Crohn disease, and ulcerative colitis. Moreover, ibrutinib and sirolimus are used for the treatment of both malignant and non-malignant diseases. The most common drug targets include ALK, B-Raf, BCR-Abl, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptor (VEGFR). Most of the small molecule inhibitors (45) interact directly with the protein kinase domain. In contrast, sirolimus, temsirolimus, and everolimus are larger molecules (MW ≈ 1000) that bind to FKBP-12 to generate a complex that inhibits mTOR (mammalian target of rapamycin). This review presents the available drug-enzyme X-ray crystal structures for 27 of the approved drugs as well as the chemical structures and physicochemical properties of all of the FDA-approved small molecule protein kinase antagonists. Six of the drugs bind covalently and irreversibly to their target. Twenty of the 48 drugs have molecular weights greater than 500, exceeding a Lipinski rule of five criterion. Excluding the macrolides (everolimus, sirolimus, temsirolimus), the average molecular weight of drugs is 480 with a range of 306 (ruxolitinib) to 615 (trametinib). Nearly half of the antagonists (23) have a lipophilic efficiency with values of less than five while the recommended optima range from 5–10. One of the vexing problems is the near universal development of resistance that is associated with the use of small molecule protein kinase inhibitors for the treatment of cancer.
Pathogenic bacteria use the type III secretion system to deliver effector proteins into host cells to modulate the host signaling pathways. In this study, the Shigella type III effector OspF was ...shown to inactivate mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases 1 and 2 (Erk1/2), c-Jun N-terminal kinase, and p38. OspF irreversibly removed phosphate groups from the phosphothreonine but not from the phosphotyrosine residue in the activation loop of MAPKs. Mass spectrometry revealed a mass loss of 98 daltons in p-Erk2, due to the abstraction of the α proton concomitant with cleavage of the C-OP bond in the phosphothreonine residue. This unexpected enzymatic activity, termed phosphothreonine lyase, appeared specific for MAPKs and was shared by other OspF family members.
Microtubule‐associated protein tau in a hyperphosphorylated state is the major component of the filamentous lesions that define a number of neurodegenerative diseases commonly referred to as ...tauopathies. Hyperphosphorylation of tau at most sites appears to precede filament assembly. Many of the hyperphosphorylated sites are serine/threonine–proline sequences. Here we show that c‐Jun N‐terminal kinases JNK1, JNK2 and JNK3 phosphorylate tau at many serine/threonine–prolines, as assessed by the generation of the epitopes of phosphorylation‐dependent anti‐tau antibodies. Of the three protein kinases, JNK2 phosphorylated the most sites in tau, followed by JNK3 and JNK1. Phosphorylation by JNK isoforms resulted in a greatly reduced ability of tau to promote microtubule assembly. These findings extend the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.
Inflammation plays a critical role in the development of diabetic cardiomyopathy (DCM), which has been identified as a major predisposing factor for heart failure in diabetic patients. Previous ...studies indicated that ivabradine (a specific agent for heart rate HR reduction) has anti‐inflammatory properties, but its role in DCM remains unknown. This study investigated whether ivabradine exerts a therapeutic effect in DCM. C57BL/6J mice were injected intraperitoneally with streptozotocin (STZ) to induce diabetes; then administered with ivabradine or saline (control). After 12 weeks, the surviving mice were analyzed to determine the cardioprotective effect of ivabradine against DCM. Although treatment with ivabradine did not affect blood glucose levels, it attenuated tumor necrosis factor‐α, interleukin‐1β, and interleukin‐6 messenger RNA (mRNA) expression, inhibited c‐Jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (p38 MAPK) activation, reduced histological abnormalities, myocardial apoptosis and collagen deposition, and improved cardiac function in the diabetic mice. Interestingly, the anti‐inflammatory and antiapoptotic properties of ivabradine, but not its inhibitory effect on JNK and p38 MAPK, were observed in high‐glucose‐cultured neonatal rat ventricular cardiomyocytes. Attenuating inflammation and apoptosis via intramyocardial injection of lentiviruses carrying short hairpin RNA targeting JNK and p38 MAPK validated that the anti‐inflammatory and antiapoptotic effects of ivabradine were partly attributed to JNK and p38 MAPK inactivation in diabetic mice. In summary, these data indicate that ivabradine‐mediated improvement of cardiac function in STZ‐induced diabetic mice may be partly attributed to inhibition of JNK/p38 MAPK‐mediated inflammation and apoptosis, which is dependent on the reduction in HR.
(a) The JNK and p38 MAPK protein levels in heart tissues after intramyocardial injection of lentiviruses carrying shRNA targeting JNK (LV‐JNK shRNA) and p38 MAPK (LV‐p38 MAPK shRNA). (b,c) Proinflammatory cytokine mRNA and protein expression were significantly decreased in diabetic mice treated with LV‐JNK shRNA and LV‐p38 MAPK shRNA. Column figure shows the difference in mRNA and protein expression. (d) TUNEL staining of heart tissues in each group.
In T lymphocytes, the mitogen-activated protein kinase (MAPK) p38 regulates pleiotropic functions and is activated by canonical MAPK signaling or the alternative activation pathway downstream of the ...T cell antigen receptor (TCR). Here we found that senescent human T cells lacked the canonical and alternative pathways for the activation of p38 but spontaneously engaged the metabolic master regulator AMPK to trigger recruitment of p38 to the scaffold protein TAB1, which caused autophosphorylation of p38. Signaling via this pathway inhibited telomerase activity, T cell proliferation and the expression of key components of the TCR signalosome. Our findings identify a previously unrecognized mode for the activation of p38 in T cells driven by intracellular changes such as low-nutrient and DNA-damage signaling (an 'intrasensory' pathway). The proliferative defect of senescent T cells was reversed by blockade of AMPK-TAB1-dependent activation of p38.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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