The GDSL Lipase MHZ11 Modulates Ethylene Signaling in Rice Roots.

Ethylene plays important roles in plant growth and development, but the regulation of ethylene signaling is largely unclear, especially in crops such as rice (Oryza sativa). Here, by analysis of the ethylene-insensitive mutant mao huzi 11 (mhz11), we identified the GDSL lipase MHZ11, which modulates ethylene signaling in rice roots. MHZ11 localized to the endoplasmic reticulum membrane and has acyl-hydrolyzing activity. This activity affects the homeostasis of sterols in rice roots and is required for root ethylene response. MHZ11 overexpression caused constitutive ethylene response in roots. Genetically, MHZ11 acts with the ethylene receptor ETHYLENE RESPONSE SENSOR2 (OsERS2) upstream of CONSTITUTIVE TRIPLE RESPONSE2 (OsCTR2) and ETHYLENE INSENSITIVE2 (OsEIN2). The mhz11 mutant maintains more OsCTR2 in the phosphorylated form whereas MHZ11 overexpression promotes ethylene-mediated inhibition of OsCTR2 phosphorylation. MHZ11 colocalized with the ethylene receptor OsERS2, and its effect on OsCTR2 phosphorylation requires ethylene perception and initiation of ethylene signaling. The mhz11 mutant overaccumulated sterols and blocking sterol biosynthesis partially rescued the mhz11 ethylene response, likely by reducing receptor-OsCTR2 interaction and OsCTR2 phosphorylation. We propose that MHZ11 reduces sterol levels to impair receptor-OsCTR2 interactions and OsCTR2 phosphorylation for triggering ethylene signaling. Our study reveals a mechanism by which MHZ11 participates in ethylene signaling for regulation of root growth in rice.

Endogenous sterol intermediates of the mevalonate pathway regulate HMGCR degradation and SREBP-2 processing.

Sterol-regulated HMG-CoA reductase (HMGCR) degradation and SREBP-2 cleavage are two major feedback regulatory mechanisms governing cholesterol biosynthesis. Reportedly, lanosterol selectively stimulates HMGCR degradation, and cholesterol is a specific regulator of SREBP-2 cleavage. However, it is unclear whether other endogenously generated sterols regulate these events. Here, we investigated the sterol intermediates from the mevalonate pathway of cholesterol biosynthesis using a CRISPR/Cas9-mediated genetic engineering approach. With a constructed HeLa cell line expressing the mevalonate transporter, we individually deleted genes encoding major enzymes in the mevalonate pathway, used lipidomics to measure sterol intermediates, and examined HMGCR and SREBP-2 statuses. We found that the C4-dimethylated sterol intermediates, including lanosterol, 24,25-dihydrolanosterol, follicular fluid meiosis activating sterol, testis meiosis activating sterol, and dihydro-testis meiosis activating sterol, were significantly upregulated upon mevalonate loading. These intermediates augmented both degradation of HMGCR and inhibition of SREBP-2 cleavage. The accumulated lanosterol induced rapid degradation of HMGCR, but did not inhibit SREBP-2 cleavage. The newly synthesized cholesterol from the mevalonate pathway is dispensable for inhibiting SREBP-2 cleavage. Together, these results suggest that lanosterol is a bona fide endogenous regulator that specifically promotes HMGCR degradation, and that other C4-dimethylated sterol intermediates may regulate both HMGCR degradation and SREBP-2 cleavage.

Neonatal Respiratory Failure with Retarded Perinatal Lung Maturation in Mice Caused by Reticulocalbin 3 Disruption.

Reticulocalbin 3 (Rcn3) is an endoplasmic reticulum lumen protein localized to the secretory pathway. As a Ca2t-binding protein of 45 kDa (Cab45)/Rcn/ER Ca2t-binding protein of 55 kDa (ERC45)/calumenin (CREC) family member, Rcn3 is reported to function as a chaperone protein involved in protein synthesis and secretion; however, the biological role of Rcn3 is largely unknown. The results presented here, for the first time, depict an indispensable physiological role of Rcn3 in perinatal lung maturation by using an Rcn3 gene knockout mouse model. These mutant mice die immediately at birth owing to atelectasis-induced neonatal respiratory distress, although these embryos are produced with grossly normal development. This respiratory distress results from a failure of functional maturation of alveolar epithelial type II cells during alveogenesis. This immaturity of type II cells is associated with a dramatic reduction in surfactant protein A and D, a disruption in surfactant phospholipid homeostasis, and a disorder in lamellar body. In vitro studies further show that Rcn3 deficiency blunts the secretion of surfactant proteins and phospholipids from lung epithelial cells, suggesting a decrease in availability of surfactants for their surface activity. Collectively, these observations indicate an essential role of Rcn3 in perinatal lung maturation and neonatal respiratory adaptation as well as shed additional light on the mechanism of neonatal respiratory distress syndrome development.

Hepatic CDP-diacylglycerol synthase 2 deficiency causes mitochondrial dysfunction and promotes rapid progression of NASH and fibrosis.

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies, ranging from steatosis to nonalcoholic steatohepatitis (NASH). The factors promoting the progression of steatosis to NASH are still unclear. Recent studies suggest that mitochondrial lipid composition is critical in NASH development. Here, we showed that CDP-DAG synthase 2 (Cds2) was downregulated in genetic or diet-induced NAFLD mouse models. Liver-specific deficiency of Cds2 provoked hepatic steatosis, inflammation and fibrosis in five-week-old mice. CDS2 is enriched in mitochondria-associated membranes (MAMs), and hepatic Cds2 deficiency impaired mitochondrial function and decreased mitochondrial PE levels. Overexpression of phosphatidylserine decarboxylase (PISD) alleviated the NASH-like phenotype in Cds2f/f;AlbCre mice and abnormal mitochondrial morphology and function caused by CDS2 deficiency in hepatocytes. Additionally, dietary supplementation with an agonist of peroxisome proliferator-activated receptor alpha (PPARα) attenuated mitochondrial defects and ameliorated the NASH-like phenotype in Cds2f/f;AlbCre mice. Finally, Cds2 overexpression protected against high-fat diet-induced hepatic steatosis and obesity. Thus, Cds2 modulates mitochondrial function and NASH development.

The Attenuation of Diabetic Nephropathy by Annexin A1 via Regulation of Lipid Metabolism Through the AMPK/PPARα/CPT1b Pathway.

Inflammation and abnormal metabolism play important roles in the pathogenesis of diabetic nephropathy (DN). Annexin A1 (ANXA1) contributes to inflammation resolution and improves metabolism. In this study, we assess the effects of ANXA1 in diabetic mice and proximal tubular epithelial cells (PTECs) treated with high glucose plus palmitate acid (HGPA) and explore the association of ANXA1 with lipid accumulation in patients with DN. It is found that ANXA1 deletion aggravates renal injuries, including albuminuria, mesangial matrix expansion, and tubulointerstitial lesions in high-fat diet/streptozotocin-induced diabetic mice. ANXA1 deficiency promotes intrarenal lipid accumulation and drives mitochondrial alterations in kidneys. In addition, Ac2-26, an ANXA1 mimetic peptide, has a therapeutic effect against lipid toxicity in diabetic mice. In HGPA-treated human PTECs, ANXA1 silencing causes FPR2/ALX-driven deleterious effects, which suppress phosphorylated Thr172 AMPK, resulting in decreased peroxisome proliferator-activated receptor α and carnitine palmitoyltransferase 1b expression and increased HGPA-induced lipid accumulation, apoptosis, and elevated expression of proinflammatory and profibrotic genes. Last but not least, the extent of lipid accumulation correlates with renal function, and the level of tubulointerstitial ANXA1 expression correlates with ectopic lipid deposition in kidneys of patients with DN. These data demonstrate that ANXA1 regulates lipid metabolism of PTECs to ameliorate disease progression; hence, it holds great potential as a therapeutic target for DN.

Targeted lipidomics reveals associations between serum sphingolipids and insulin sensitivity measured by the hyperinsulinemic-euglycemic clamp.

AIMS: Sphingolipids(SPs) and their substrates and constituents, fatty acids (FAs), are implicated in the pathogenesis of various metabolic diseases associated. This study aimed to systematically investigate the associations between serum sphingolipids and insulin sensitivity as well as insulin secretion. METHODS: We conducted a lipidomics evaluation of molecularly distinct SPs in the serum of 86 consecutive Chinese adults using LC/MS. The glucose infusion rate over 30 min (GIR30) was measured under steady conditions to assess insulin sensitivity by the gold standard hyperinsulinemic-euglycemic clamp. We created the ROC curves to detect the serum SMs diagnostic value. RESULTS: Total and subspecies of serum SMs and globotriaosyl ceramides (Gb3s) were positively related to GIR30, free FAs (FFA 16:1, FFA20:4), some long chain GM3 and complex ceramide GluCers showed strong negative correlations with GIR30. Notably, ROC curves showed that SM/Cer and SM d18:0/26:0 may be good serum lipid predictors of diagnostic indicators of insulin sensitivity close to conventional clinical indexes such as 1/HOMA-IR (areas under the curve > 0.80) based on GIR30 as standard diagnostic criteria, and (SM/Cer)/(BMI*LDLc) areas under the curve = 0.93) is the best. CONCLUSIONS: These results provide novel associations between serum sphingolipid between insulin sensitivity measured by the hyperinsulinemic-euglycemic clamp and identify two specific SPs that may represent prognostic biomarkers for insulin sensitivity.

Noncanonical regulation of phosphatidylserine metabolism by a Sec14-like protein and a lipid kinase.

The yeast phosphatidylserine (PtdSer) decarboxylase Psd2 is proposed to engage in a membrane contact site (MCS) for PtdSer decarboxylation to phosphatidylethanolamine (PtdEtn). This proposed MCS harbors Psd2, the Sec14-like phosphatidylinositol transfer protein (PITP) Sfh4, the Stt4 phosphatidylinositol (PtdIns) 4-OH kinase, the Scs2 tether, and an uncharacterized protein. We report that, of these components, only Sfh4 and Stt4 regulate Psd2 activity in vivo. They do so via distinct mechanisms. Sfh4 operates via a mechanism for which its PtdIns-transfer activity is dispensable but requires an Sfh4-Psd2 physical interaction. The other requires Stt4-mediated production of PtdIns-4-phosphate (PtdIns4P), where Stt4 (along with the Sac1 PtdIns4P phosphatase and endoplasmic reticulum-plasma membrane tethers) indirectly modulate Psd2 activity via a PtdIns4P homeostatic mechanism that influences PtdSer accessibility to Psd2. These results identify an example in which the biological function of a Sec14-like PITP is cleanly uncoupled from its canonical in vitro PtdIns-transfer activity and challenge popular functional assumptions regarding lipid-transfer protein involvements in MCS function.

Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth.

VAMP7 is involved in autophagy and in exocytosis-mediated neurite growth, two yet unconnected cellular pathways. Here, we find that nutrient restriction and activation of autophagy stimulate axonal growth, while autophagy inhibition leads to loss of neuronal polarity. VAMP7 knockout (KO) neuronal cells show impaired neurite growth, whereas this process is increased in autophagy-null ATG5 KO cells. We find that endoplasmic reticulum (ER)-phagy-related LC3-interacting-region-containing proteins Atlastin 3 and Reticulon 3 (RTN3) are more abundant in autophagy-related protein ATG5 KO and less abundant in VAMP7 KO secretomes. Treatment of neuronal cells with ATG5 or VAMP7 KO conditioned medium does not recapitulate the effect of these KOs on neurite growth. A nanobody directed against VAMP7 inhibits axonal overgrowth induced by nutrient restriction. Furthermore, expression of the inhibitory Longin domain of VAMP7 impairs the subcellular localization of RTN3 in neurons. We propose that VAMP7-dependent secretion of RTN3 regulates neurite growth.

An electron transfer path connects subunits of a mycobacterial respiratory supercomplex.

We report a 3.5-angstrom-resolution cryo-electron microscopy structure of a respiratory supercomplex isolated from Mycobacterium smegmatis. It comprises a complex III dimer flanked on either side by individual complex IV subunits. Complex III and IV associate so that electrons can be transferred from quinol in complex III to the oxygen reduction center in complex IV by way of a bridging cytochrome subunit. We observed a superoxide dismutase-like subunit at the periplasmic face, which may be responsible for detoxification of superoxide formed by complex III. The structure reveals features of an established drug target and provides a foundation for the development of treatments for human tuberculosis.