This study assessed the association of pathological kidney lesions with cardiovascular events in biopsy-proven diabetic kidney disease (DKD) with type 2 diabetes. This multicenter, retrospective study involved 244 patients with no previous cardiovascular events before biopsy, estimated glomerular filtration rate (eGFR) ≥ 30 mL/min/1.73 m2 at biopsy (baseline), and ≥ 1 year of observation after biopsy. The outcomes were the first occurrence of cardiovascular events (cardiovascular death, non-fatal myocardial infarction, coronary intervention, or non-fatal stroke), and non-cardiovascular deaths before cardiovascular events were considered competing events. The association between the severity of each pathological lesion and cardiovascular events was investigated. During follow-up (median: 6.4 years), 43 patients experienced cardiovascular events. The baseline clinical characteristics did not differ according to cardiovascular events. The cumulative incidence of cardiovascular events was higher in patients with mesangiolysis, global glomerulosclerosis ≥ 50

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain of glycerophospholipids. Polyunsaturated fatty acids (PUFAs) are preferentially attached at the sn-2 position of glycerophospholipids and are easily truncated by oxidation. The truncated-oxidized phospholipids (tr-oxPLs) trigger various cellular responses, and PLA2s may play a critical role in the metabolism of the tr-oxPLs by removing the oxidized sn-2 chain. In the present study, we demonstrated using an in vitro lipidomics assay that Group VIA calcium-independent PLA2 (GVIA iPLA2) showed high activity toward phosphatidylcholine with a 9-oxononanoyl chain, but not with an azelaoyl chain on the sn-2 position. We conducted molecular dynamics simulations which revealed that the hydrophilicity of the sn-2 acyl chain critically affects the binding of the substrate in the active site. Based on the unique specificity of GVIA iPLA2 toward tr-oxPLs, we synthesized an oxidatively modified inhibitor (GK766) for GVIA iPLA2, aiming for improvement of its selectivity and/or potency. As we expected, the modified inhibitor improved its selectivity of GVIA iPLA2 compared to the unmodified inhibitor (GK187), although the inhibitory effect became somewhat weaker. More importantly, we demonstrated that GK766 induces cell death by ferroptosis more effectively than GK187 using an erythroleukemia cell line. In the present study, we have further defined the unique substrate specificity of GVIA iPLA2 toward tr-oxPLs and its molecular mechanism. Furthermore, we have developed a novel specificity-based inhibitor that induces ferroptosis demonstrating that using substrate selectivity helps in developing more effective therapeutics.

Clostridioides difficile infection (CDI) is a gastrointestinal condition that arises from the disruption of normal intestinal flora, often caused by antibiotics or antiacids. Fulminant CDI, characterized by shock, intestinal obstruction, and toxic megacolon, has a poor prognosis. Patients with chronic kidney disease and those on maintenance hemodialysis are at a higher risk for developing CDI, experiencing severe disease progression, and facing increased mortality. A 70 years-old man with diabetic nephropathy undergoing maintenance hemodialysis for 2 years developed fulminant CDI. He had been on long-term antibiotic therapy for chronic pyogenic knee arthritis for 5 months. He presented with severe watery diarrhea, and stool tests confirmed CD toxins A and B. A computed tomography (CT) scan revealed toxic megacolon with pronounced intestinal edema, bowel dilatation, and massive pleural effusion. He was in shock and admitted to the intensive care unit. Treatment included massive fluid infusion, continuous vasopressor support, and regular human albumin infusions to stabilize circulation. Antibiotic therapy was initiated with oral vancomycin (2000 mg/day) and intravenous metronidazole (1500 mg/day). Advanced supportive measures included polymyxin B-immobilized fiber column direct hemoperfusion (PMX-DHP) and continuous renal replacement therapy using a cytokine adsorption column (SepXiris®). The patient’s condition gradually improved, and antimicrobial therapy was completed on day 25 of hospitalization. Follow-up computed tomography (CT) scans showed resolution of toxic megacolon, and there were no signs of CDI recurrence. The patient was eventually transferred to a rehabilitation facility. Despite established treatments, fulminant CDI carries a high mortality rate, particularly in patients on maintenance hemodialysis, where fatal outcomes are common. This case highlights the successful management of fulminant CDI in a hemodialysis patient through a multitargeted approach, including intensive care, circulatory support with albumin monitoring, and advanced therapies such as PMX-DHP and cytokine adsorption. This report underscores the importance of comprehensive strategies in improving outcomes for high-risk patients.

Eicosanoids are key players in inflammatory diseases and cancer. Targeting their production by inhibiting Group IVA cytosolic phospholipase A2 (cPLA2α) offers a promising approach for cancer therapy. In this study, we synthesize a second generation of thiazolyl ketone inhibitors of cPLA2α starting with compound GK470 (AVX235) and test their in vitro and cellular activities. We identify a more potent and selective lead molecule, GK420 (AVX420), which we test in parallel with AVX235 and a structurally unrelated compound, AVX002 for inhibition of cell viability across a panel of cancer cell lines. From this, we show that activity of polycomb group repressive complex 2 is a key molecular determinant of sensitivity to cPLA2α inhibition, while resistance depends on antioxidant response pathways. Consistent with these results, we show that elevated intracellular reactive oxygen species and activating transcription factor 4 target gene expression precede cell death in AVX420-sensitive T-cell acute lymphoblastic leukemia cells. Our findings imply cPLA2α may support cancer by mitigating oxidative stress and inhibiting tumor suppressor expression and suggest that AVX420 has potential for treating acute leukemias and other cancers that are susceptible to oxidative cell death.

Fibrosis is a common end-stage pathway of progressive chronic kidney diseases. Previously we demonstrated that myocardin-related transcription factor (MRTF)-serum response factor (SRF) signaling drives the expression of fibrosis-related molecules through actin cytoskeleton dynamics in renal fibroblasts. However, it has not been elucidated whether actin-associated proteins relate to the pathogenesis of fibrosis. Here, we reveal that the actin cytoskeleton-regulating pathway is significantly correlated with estimated glomerular filtration rate (eGFR) and collagen type 1 alpha 1 expression in human proteome analysis. We found that palladin was one of the TGF-β1-dependent actin-associated proteins in renal fibroblasts. Our mechanistic studies demonstrated that palladin activates MRTF-SRF signaling via actin cytoskeleton rearrangement upon TGF-β1 stimulation. In addition, palladin expression itself was enhanced by MRTF-SRF signaling, indicating a positive feedback loop. In vitro, genetic silencing of the palladin-MRTF-SRF axis suppressed extracellular matrix production and myofibroblast differentiation. In preclinical models in vivo, fibroblast-specific palladin-deficient mice (palladiniFBKO) were protected from kidney dysfunction and fibrosis that developed in adenine-induced nephropathy, which was associated with reduced numbers of myofibroblasts compared to wild type (palladinF/F) mice. In patients with renal disease, palladin was significantly upregulated in the renal interstitium of patients with low eGFR and kidney fibrosis. Moreover, upregulation of the palladin-MRTF-SRF axis correlated with kidney function and fibrosis in patients with various kidney diseases, including IgA nephropathy, diabetic nephropathy, and nephrosclerosis. Taken together, we consider palladin to be a novel regulator of actin cytoskeleton signaling in fibrotic fibroblasts and represents a novel therapeutic target for the treatment of progressive kidney diseases. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

AIM:Fibrosis is a common mechanism underlying the progression of kidney and other organ failures. Complement protein C1q and chemokine receptor 8 (CCR8), whose ligand is CCL1, are implicated in fibrosis. This study aimed to evaluate the clinical significance of CD45+ mononuclear cells coexpressing C1q and CCR8 (CD45 + C1q + CCR8+ cells) in kidney disease. METHODS:This prospective observational study included 44 patients with kidney disease. The percentage of CD45 + C1q + CCR8+ cells among CD45+ mononuclear cells in the peripheral blood was measured using flow cytometry at baseline. Correlations between these percentages and clinical parameters, including serum creatinine (Cr) and urinary protein levels, were examined using linear regression models. The association between baseline percentages of CD45 + C1q + CCR8+ cells and kidney outcomes-defined as end-stage kidney disease or a 30% decrease in the estimated glomerular filtration rate-was examined using Cox proportional hazards models in patients with serum Cr data during follow-up. RESULTS:The median age of the cohort was 57 years, and 61.4% were male. The median serum Cr and urinary protein levels were 1.73 mg/dL and 1.99 g/g Cr, respectively. Baseline percentages of CD45 + C1q + CCR8+ cells positively correlated with serum Cr (p = 0.028) and urinary protein (p = 0.015). Among 21 patients with follow-up data, 10 (48%) reached kidney outcomes. Patients with moderately elevated percentages had a higher risk of kidney outcomes than those with low levels (HR: 27.31, 95% CI: 1.08-692.3; p = 0.04). CONCLUSION:CD45 + C1q + CCR8+ cell percentages in peripheral blood may reflect kidney function and are associated with disease progression, indicating their potential as biomarkers.

Background: Diabetes is a significant social issue. Controlling diabetic complications such as nephropathy is crucial for the quality of life (QOL) of diabetic patients. The abnormal activation of protein kinase C (PKC) through increased diacylglycerol levels (DG) due to hyperglycemia is a common mechanism that causes diabetic complications. Diacylglycerol kinase (DGK) can attenuate PKC activity by converting DG to phosphatidic acid. Thus far, d-α-tocopherol (αToc) treatment has been shown to prevent early changes of diabetic renal dysfunctions by activating DGKα via the 67KDa laminin receptor. Vitamin E is classified into tocopherols (Toc) and tocotrienols (T3) with four derivatives: α, β, γα, β, γ, and δ. We have found that all Tocs induced the translocation of DGKα. However, it remains unclear if T3s also induced translocation of DGKα. Objective: The objective of the study was to investigate the relationship between tocotrienols (T3s) and induced translocation of DGKα, while exploring the mechanism underlying T3s-induced translocation of DGKα. Results: Similarly to a-tocopherol, α-T3 binds to 67LR, resulting in the induction of a translocation of DGK. As well as α- T3, β, γ, δ, and α Tocs induced translocation of DGK. Conclusion: αT3, like αToc, activates DGKα by binding to Leu58 and Trp176 in 67LR. It means 67LR may be important for the functions of T3s, especially the non-oxidant effects of T3s. Keywords: catechins, diabetic nephropathy, diacylglycerol kinase, vitamin E

Introduction: Peritoneal equilibration test (PET) has been used to monitor peritoneal function. A more convenient marker would be useful in clinical situations including home medical care. Autotaxin is known to leak into the interstitium as vascular permeability increases during the progression of tissue fibrosis. Therefore, we hypothesized that autotaxin concentrations in peritoneal dialysis (PD) effluent might reflect peritoneal function. Methods: This study enrolled 45 patients undergoing PD from 2016 to 2021. Autotaxin concentrations measured in PD effluent were evaluated for their associations with markers obtained from PET. Results: Mean age was 69 years, and 33 patients were men. Univariate and multivariate analyses revealed that autotaxin concentrations are associated with dialysate/plasma creatinine ratio, end/start dialysate glucose ratio, and the dip in the dialysate sodium concentration, a marker of ultrafiltration capacity, at baseline (all p < 0.05). Conclusions: Autotaxin concentrations in PD effluent might be an adjunct marker that reflects peritoneal function.

AIM:Recently, substantial studies have been accumulated to indicate the important role of gut microbiota in diabetic kidney disease (DKD). The abnormal change of bacterial-derived products could imply specific injuries or play beneficial or harmful roles in DKD progression. In this study, we examined the presence and contribution of the Klebsiella oxytoca gene in the circulation of patients with DKD. METHOD:We enrolled a total of 16 healthy participants, 17 patients with DKD, 5 patients with DKD requiring haemodialysis (HD), and 7 patients with CKD without diabetes. Bacterial-derived DNA (16S rDNA and a specific K. oxytoca gene) in the blood was detected using droplet digital PCR, then investigated the relationship with clinical characteristics. RESULTS:We identified an increase in K. oxytoca genes in the blood of DKD patients. Interestingly, blood K. oxytoca copies and K. oxytoca/ 16S DNA ratio correlated with higher blood creatinine and BUN levels together with lower eGFR in DKD patients. K. oxytoca levels were also associated with higher neutrophil percentage, lower lymphocyte frequency, and increased neutrophil-to-lymphocyte ratio. CONCLUSION:Collectively, the presence of the K. oxytoca gene in the circulation could serve as a biomarker reflecting reduced renal function in DKD patients.

Background: D-alanine administration prevented kidney damage in a murine acute kidney injury model. Further data are needed on the influence of D-alanine on kidney function in humans. Objective: This study investigated the effects of D-alanine intake on amino acid metabolism and kidney function in healthy volunteers. Methods: This multicenter pilot study randomly assigned individuals from the general Japanese population to receive 3 g or 6 g of D-alanine intake per day for 7 d in a 1:1 ratio. The primary endpoint was the mean change in plasma and urine D-alanine levels from baseline to 7 d after intake. The secondary endpoints were mean changes in kidney function and other clinical factors. Safety was assessed by evaluating adverse events and clinical parameters. Results: We randomly assigned 24 participants to the 3-g (n = 12) and 6-g D-alanine (n = 12) groups. The mean baseline estimated glomerular filtration rate (eGFR) was 73 mL/min/1.73 m2. 2 . The mean plasma D-alanine concentration increased from baseline by 77.5 +/- 34.3 and 192.1 +/- 80.9 nmol/mL in the 3-g and 6-g D-alanine groups (both p < 0.0001), respectively, in a dose-dependent manner (between- group difference: 114.6 nmol/mL; 95% CI: 62.1-167.2; - 167.2; P = 0.0002). A similar increase was observed for the urine D-alanine to creatinine ratio. The mean eGFR was elevated by 5.7 f 8.8 mL/min/1.73 m(2) in the 6-g D-alanine group (P = 0.045) but did not significantly change in the 3-g D-alanine group. Nonserious adverse events were reported in 11 participants. Conclusions: D-alanine intake increased plasma and urine D-alanine levels and was well tolerated in participants with normal kidney function. These results will be useful in future trials investigating the effects of D-alanine intake on kidney disease progression in patients with chronic kidney disease. This trial was registered at the UMIN Clinical Trials Registry as UMIN000051466.

Phospholipase A2's (PLA2's) constitute a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain on glycerophospholipids. We have previously reported that each PLA2 Type shows a unique substrate specificity for the molecular species it hydrolyzes, especially the acyl chain that is cleaved from the sn-2 position and to some extent the polar group. However, phosphatidylinositol (PI) and PI phosphates (PIPs) have not been as well studied as substrates as other phospholipids because the PIPs require adaptation of the standard analysis methods, but they are important in vivo. We determined the in vitro activity of the three major types of human PLA2's, namely the cytosolic (c), calcium-independent (i), and secreted (s) PLA2's toward PI, PI-4-phosphate (PI(4)P), and PI-4,5-bisphosphate (PI(4,5)P2). The in vitro assay revealed that Group IVA cPLA2 (GIVA cPLA2) showed relatively high activity toward PI and PI(4)P among the tested PLA2's; nevertheless, the highly hydrophilic headgroup disrupted the interaction between the lipid surface and the enzyme. GIVA cPLA2 and GVIA iPLA2 showed detectable activity toward PI(4,5)P2, but it appeared to be a poorer substrate for all of the PLA2's tested. Furthermore, molecular dynamics (MD) simulations demonstrated that Thr416 and Glu418 of GIVA cPLA2 contribute significantly to accommodating the hydrophilic head groups of PI and PI(4)P, which could explain some selectivity for PI and PI(4)P. These results indicated that GIVA cPLA2 can accommodate PI and PI(4)P in its active site and hydrolyze them, suggesting that the GIVA cPLA2 may best account for the PI and PIP hydrolysis in living cells.

Group VIA calcium- independent phospholipase A2 (iPLA2) is a member of the PLA2 superfamily that exhibits calcium- independent activity in contrast to the other two major types, secreted phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2), which both require calcium for their enzymatic activity. Adenosine triphosphate (ATP) has been reported to allosterically activate iPLA2, and this has now been verified with a lipidomics-based mixed- micelle assay, but its mechanism of action has been unknown. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed to identify ATP interaction peptide regions located within the ankyrin repeat domain at which ATP interacts. Molecular dynamics simulations revealed the mechanism bywhich ATP binds to its site and the main residues that interact. Site- directed mutagenesis was used to verify the importance of these residues in the role of ATP in regulating iPLA2 activity. Importantly, calcium was found to abolish the enhancing regulatory function of ATP and to promote the inhibitory activity by calmodulin. Given previous evidence that calcium does not bind directly to iPLA2, its effect appears to be indirect via association with ATP and/or calmodulin. Using HDX-MS, we found that calmodulin interacts with the N terminus peptide region of iPLA2 consisting of residues 20 to 28. These two regulatory iPLA2 sites open the road to the development of potential targets for therapeutic intervention.

Despite treatment advances, acute kidney injury (AKI)-related mortality rates are still high in hospitalized adults, often due to sepsis. Sepsis and AKI could synergistically worsen the outcomes of critically ill patients. TLR4 signaling and mitochondrial antiviral signaling protein (MAVS) signaling are innate immune responses essential in kidney diseases, but their involvement in sepsis-associated AKI (SA-AKI) remains unclear. We studied the role of MAVS in kidney injury related to the TLR4 signaling pathway using a murine LPS-induced AKI model in wild-type and MAVS-knockout mice. We confirmed the importance of M1 macrophage in SA-AKI through in vivo assessment of inflammatory responses. The TLR4 signaling pathway was upregulated in activated bone marrow-derived macrophages, in which MAVS helped maintain the LPS-suppressed TLR4 mRNA level. MAVS regulated redox homeostasis via NADPH oxidase Nox2 and mitochondrial reverse electron transport in macrophages to alleviate the TLR4 signaling response to LPS. Hypoxia-inducible factor 1α (HIF-1α) and AP-1 were key regulators of TLR4 transcription and connected MAVS-dependent reactive oxygen species signaling with the TLR4 pathway. Inhibition of succinate dehydrogenase could partly reduce inflammation in LPS-treated bone marrow-derived macrophages without MAVS. These findings highlight the renoprotective role of MAVS in LPS-induced AKI by regulating reactive oxygen species generation-related genes and maintaining redox balance. Controlling redox homeostasis through MAVS signaling may be a promising therapy for SA-AKI.

Alzheimer's disease is a neurologic disorder characterized by the accumulation of extracellular deposits of amyloid-β (Aβ) fibrils in the brain of patients. The key etiologic agent in Alzheimer's disease is not known; however oligomeric Aβ appears detrimental to neuronal functions and increases Aβ fibrils deposition. Previous research has shown that curcumin, a phenolic pigment of turmeric, has an effect on Aβ assemblies, although the mechanism remains unclear. In this study, we demonstrate that curcumin disassembles pentameric oligomers made from synthetic Aβ42 peptides (pentameric oAβ42), using atomic force microscopy imaging followed by Gaussian analysis. Since curcumin shows keto-enol structural isomerism (tautomerism), the effect of keto-enol tautomerism on its disassembly was investigated. We have found that curcumin derivatives capable of keto-enol tautomerization also disassemble pentameric oAβ42, while, a curcumin derivative incapable of tautomerization did not affect the integrity of pentameric oAβ42. These experimental findings indicate that keto-enol tautomerism plays an essential role in the disassembly. We propose a mechanism for oAβ42 disassembly by curcumin based on molecular dynamics calculations of the tautomerism. When curcumin and its derivatives bind to the hydrophobic regions of oAβ42, the keto-form changes predominantly to the enol-form; this transition is associated with structural (twisting, planarization and rigidification) and potential energy changes that give curcumin enough force to act as a torsion molecular-spring that eventually disassembles pentameric oAβ42. This proposed mechanism sheds new light on keto-enol tautomerism as a relevant chemical feature for designing such novel therapeutic drugs that target protein aggregation.

Glycerophospholipids are major components of cell membranes and consist of a glycerol backbone esterified with one of over 30 unique fatty acids at each of the sn-1 and sn-2 positions. In addition, in some human cells and tissues as much as 20% of the glycerophospholipids contain a fatty alcohol rather than an ester in the sn-1 position, although it can also occur in the sn-2 position. The sn-3 position of the glycerol backbone contains a phosphodiester bond linked to one of more than 10 unique polar head-groups. Hence, humans contain thousands of unique individual molecular species of phospholipids given the heterogeneity of the sn-1 and sn-2 linkage and carbon chains and the sn-3 polar groups. Phospholipase A2 (PLA2) is a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain resulting in lyso-phospholipids and free fatty acids that then undergo further metabolism. PLA2's play a critical role in lipid-mediated biological responses and membrane phospholipid remodeling. Among the PLA2 enzymes, the Group VIA calcium-independent PLA2 (GVIA iPLA2), also referred to as PNPLA9, is a fascinating enzyme with broad substrate specificity and it is implicated in a wide variety of diseases. Especially notable, the GVIA iPLA2 is implicated in the sequelae of several neurodegenerative diseases termed "phospholipase A2-associated neurodegeneration" (PLAN) diseases. Despite many reports on the physiological role of the GVIA iPLA2, the molecular basis of its enzymatic specificity was unclear. Recently, we employed state-of-the-art lipidomics and molecular dynamics techniques to elucidate the detailed molecular basis of its substrate specificity and regulation. In this review, we summarize the molecular basis of the enzymatic action of GVIA iPLA2 and provide a perspective on future therapeutic strategies for PLAN diseases targeting GVIA iPLA2.

Fibroblast accumulation and extracellular matrix (ECM) deposition are common critical steps for the progression of organ fibrosis, but the precise molecular mechanisms remain to be fully investigated. We have previously demonstrated that lysophosphatidic acid contributes to organ fibrosis through the production of connective tissue growth factor (CTGF) via actin cytoskeleton‐dependent signaling, myocardin‐related transcription factor family (MRTF) consisting of MRTF‐A and MRTF‐B‐serum response factor (SRF) pathway. In this study, we investigated the role of the MRTF‐SRF pathway in the development of renal fibrosis, focusing on the regulation of ECM‐focal adhesions (FA) in renal fibroblasts. Here we showed that both MRTF‐A and ‐B were required for the expressions of ECM‐related molecules such as lysyl oxidase family members, type I procollagen and fibronectin in response to transforming growth factor (TGF)‐β1. TGF‐β1‐MRTF‐SRF pathway induced the expressions of various components of FA such as integrin α subunits (αv, α2, α11) and β subunits (β1, β3, β5) as well as integrin‐linked kinase (ILK). On the other hand, the blockade of ILK suppressed TGF‐β1‐induced MRTF‐SRF transcriptional activity, indicating a mutual relationship between MRTF‐SRF and FA. Myofibroblast differentiation along with CTGF expression was also dependent on MRTF‐SRF and FA components. Finally, global MRTF‐A deficient and inducible fibroblast‐specific MRTF‐B deficient mice (MRTF‐AKOBiFBKO mice) are protected from renal fibrosis with adenine administration. Renal expressions of ECM‐FA components and CTGF as well as myofibroblast accumulation were suppressed in MRTF‐AKOBiFBKO mice. These results suggest that the MRTF‐SRF pathway might be a therapeutic target for renal fibrosis through the regulation of components forming ECM‐FA in fibroblasts.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein's phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA2 binding to a phospholipid surface. This allosteric regulation of an enzyme's activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA2 and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the sn-2 position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA2's specificity toward oxidized phospholipids.

It is generally recognized that the main function of α-tocopherol (αToc), which is the most active form of vitamin E, is its antioxidant effect, while non-antioxidant effects have also been reported. We previously found that αToc ameliorates diabetic nephropathy via diacylglycerol kinase alpha (DGKα) activation in vivo, and the activation was not related to the antioxidant effect. However, the underlying mechanism of how αToc activates DGKα have been enigmatic. We report that the membrane-bound 67 kDa laminin receptor (67LR), which has previously been shown to serve as a receptor for epigallocatechin gallate (EGCG), also contains a novel binding site for vitamin E, and its association with Vitamin E mediates DGKα activation by αToc. We employed hydrogen-deuterium exchange mass spectrometry (HDX/MS) and molecular dynamics (MD) simulations to identify the specific binding site of αToc on the 67LR and discovered the conformation of the specific hydrophobic pocket that accommodates αToc. Also, HDX/MS and MD simulations demonstrated the detailed binding of EGCG to a water-exposed hydrophilic site on 67LR, while in contrast αToc binds to a distinct hydrophobic site. We demonstrated that 67LR triggers an important signaling pathway mediating non-antioxidant effects of αToc, such as DGKα activation. This is the first evidence demonstrating a membrane receptor for αToc and one of the underlying mechanisms of a non-antioxidant function for αToc.

The quest for novel agents to regulate the generation of prostaglandin E2 (PGE2) is of high importance because this eicosanoid is a key player in inflammatory diseases. We synthesized a series of N-acylated and N-alkylated 2-aminobenzothiazoles and related heterocycles (benzoxazoles and benzimidazoles) and evaluated their ability to suppress the cytokine-stimulated generation of PGE2 in rat mesangial cells. 2-Aminobenzothiazoles, either acylated by the 3-(naphthalen-2-yl)propanoyl moiety (GK510) or N-alkylated by a chain carrying a naphthalene (GK543) or a phenyl moiety (GK562) at a distance of three carbon atoms, stand out in inhibiting PGE2 generation, with EC50 values ranging from 118 nM to 177 nM. Both GK510 and GK543 exhibit in vivo anti-inflammatory activity greater than that of indomethacin. Thus, N-acylated or N-alkylated 2-aminobenzothiazoles are novel leads for the regulation of PGE2 formation.

Glycerophospholipids are major components of cell membranes and have enormous variation in the composition of fatty acyl chains esterified on the sn-1 and sn-2 position as well as the polar head groups on the sn-3 position of the glycerol backbone. Phospholipase A2 (PLA2) enzymes constitute a superfamily of enzymes which play a critical role in metabolism and signal transduction by hydrolyzing the sn-2 acyl chains of glycerophospholipids. In human cell membranes, in addition to the conventional diester phospholipids, a significant amount is the sn-1 ether-linked phospholipids which play a critical role in numerous biological activities. However, precisely how PLA2s distinguish the sn-1 acyl chain linkage is not understood. In the present study, we expanded the technique of lipidomics to determine the unique in vitro specificity of three major human PLA2s, including Group IVA cytosolic cPLA2, Group VIA calcium-independent iPLA2, and Group V secreted sPLA2 toward the linkage at the sn-1 position. Interestingly, cPLA2 prefers sn-1 vinyl ether phospholipids known as plasmalogens over conventional ester phospholipids and the sn-1 alkyl ether phospholipids. iPLA2 showed similar activity toward vinyl ether and ester phospholipids at the sn-1 position. Surprisingly, sPLA2 preferred ester phospholipids over alkyl and vinyl ether phospholipids. By taking advantage of molecular dynamics simulations, we found that Trp30 in the sPLA2 active site dominates its specificity for diester phospholipids.