The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that ...traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterol
like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export.
Degradation of endoplasmic reticulum (ER) by selective autophagy (ER‐phagy) is crucial for ER homeostasis. However, it remains unclear how ER scission is regulated for subsequent autophagosomal ...sequestration and lysosomal degradation. Here, we show that oligomerization of ER‐phagy receptor FAM134B (also referred to as reticulophagy regulator 1 or RETREG1) through its reticulon‐homology domain is required for membrane fragmentation in vitro and ER‐phagy in vivo. Under ER‐stress conditions, activated CAMK2B phosphorylates the reticulon‐homology domain of FAM134B, which enhances FAM134B oligomerization and activity in membrane fragmentation to accommodate high demand for ER‐phagy. Unexpectedly, FAM134B G216R, a variant derived from a type II hereditary sensory and autonomic neuropathy (HSAN) patient, exhibits gain‐of‐function defects, such as hyperactive self‐association and membrane scission, which results in excessive ER‐phagy and sensory neuron death. Therefore, this study reveals a mechanism of ER membrane fragmentation in ER‐phagy, along with a signaling pathway in regulating ER turnover, and suggests a potential implication of excessive selective autophagy in human diseases.
Synopsis
How endoplasmic reticulum (ER) membranes are fragmented for subsequent autophagic degradation (ER‐phagy) is ill‐defined. CAMK2B‐dependent phosphorylation of ER‐phagy receptor FAM134B promotes its oligomerization and membrane scission activity, a process deregulated in sensory neuropathy.
ER‐phagy receptor FAM134B oligomerizes through its reticulon‐homology domain (RHD).
FAM134B oligomerization is required for ER membrane scission prior to autophagosomal engulfment.
ER stress triggers the activation of CAMK2B, which phosphorylates FAM134B to enhance ER membrane fragmentation and ER‐phagy.
An HSAN type‐II patient‐derived variant, FAM134B‐G216R, forms higher‐order oligomers and induces massive ER‐phagy, which leads to sensory neuron death.
CAMK2B‐dependent phosphorylation of autophagy receptor FAM134B promotes its oligomerization and membrane‐scission activity, a process deregulated in sensory neuropathy.
The nucleotide oligomerization domain (NOD)-like receptors 1 and 2 (NOD1/2) are intracellular pattern-recognition proteins that activate immune signaling pathways in response to peptidoglycans ...associated with microorganisms. Recruitment to bacteria-containing endosomes and other intracellular membranes is required for NOD1/2 signaling, and NOD1/2 mutations that disrupt membrane localization are associated with inflammatory bowel disease and other inflammatory conditions. However, little is known about this recruitment process. We found that NOD1/2 S-palmitoylation is required for membrane recruitment and immune signaling. ZDHHC5 was identified as the palmitoyltransferase responsible for this critical posttranslational modification, and several disease-associated mutations in NOD2 were found to be associated with defective S-palmitoylation. Thus, ZDHHC5-mediated S-palmitoylation of NOD1/2 is critical for their ability to respond to peptidoglycans and to mount an effective immune response.
Macroautophagy (autophagy) utilizes a serial of receptors to specifically recognize and degrade autophagy cargoes, including damaged organelles, to maintain cellular homeostasis. Upstream signals ...spatiotemporally regulate the biological functions of selective autophagy receptors through protein post-translational modifications (PTM) such as phosphorylation. However, it is unclear how acetylation directly controls autophagy receptors in selective autophagy. Here, we report that an ER-phagy receptor FAM134B is acetylated by CBP acetyltransferase, eliciting intense ER-phagy. Furthermore, FAM134B acetylation promoted CAMKII-mediated phosphorylation to sustain a mode of milder ER-phagy. Conversely, SIRT7 deacetylated FAM134B to temper its activities in ER-phagy to avoid excessive ER degradation. Together, this work provides further mechanistic insights into how ER-phagy receptor perceives environmental signals for fine-tuning of ER homeostasis and demonstrates how nucleus-derived factors are programmed to control ER stress by modulating ER-phagy.
The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from ...mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo. Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.
Trained immunity, induced by β-glucan in monocytes, is mediated by activating metabolic pathways that result in epigenetic rewiring of cellular functional programs; however, molecular mechanisms ...underlying these changes remain unclear. Here, we report a key immunometabolic and epigenetic pathway mediated by the miR-9-5p-isocitrate dehydrogenase 3α (IDH3α) axis in trained immunity. We found that β-glucan-trained miR-9-5p-/- monocytes showed decreased IL-1β, IL-6, and TNF-α production after LPS stimulation. Trained miR-9-5p-/- mice produced decreased levels of proinflammatory cytokines upon rechallenge in vivo and had worse protection against Candida albicans infection. miR-9-5p targeted IDH3α and reduced α-ketoglutarate (α-KG) levels to stabilize HIF-1α, which promoted glycolysis. Accumulating succinate and fumarate via miR-9-5p action integrated immunometabolic circuits to induce histone modifications by inhibiting KDM5 demethylases. β-Glucan-trained monocytes exhibited low IDH3α levels, and IDH3α overexpression blocked the induction of trained immunity by monocytes. Monocytes with IDH3α variants from autosomal recessive retinitis pigmentosa patients showed a trained immunity phenotype at immunometabolic and epigenetic levels. These findings suggest that miR-9-5p and IDH3α act as critical metabolic and epigenetic switches in trained immunity.
The aim of this study was to test whether a combined risk score on the basis of genetic risk and serology can improve the prediction of kidney failure in phospholipase A2 receptor (PLA2R)-associated ...primary membranous nephropathy.
We performed a retrospective analysis of 519 biopsy-proven PLA2R-associated primary membranous nephropathy patients with baseline eGFR ≥25 ml/min per 1.73 m 2 . The combined risk score was calculated by combining the genetic risk score with PLA2R ELISA antibody titers. The primary end point was kidney disease progression defined as a 50% reduction in eGFR or kidney failure. Cox proportional hazard regression analysis and C-statistics were applied to compare the performance of PLA2R antibody, genetic risk score, and combined risk score, as compared with clinical factors alone, in predicting primary outcomes.
The median age was 56 years (range, 15-82 years); the male-to-female ratio was 1:0.6, the median eGFR at biopsy was 99 ml/min per 1.73 m 2 (range: 26-167 ml/min per 1.73 m 2 ), and the median proteinuria was 5.3 g/24 hours (range: 1.5-25.8 g/24 hours). During a median follow-up of 67 (5-200) months, 66 (13%) had kidney disease progression. In Cox proportional hazard regression models, PLA2R antibody titers, genetic risk score, and combined risk score were all individually associated with kidney disease progression with and without adjustments for age, sex, proteinuria, eGFR, and tubulointerstitial lesions. The best-performing clinical model to predict kidney disease progression included age, eGFR, proteinuria, serum albumin, diabetes, and tubulointerstitial lesions (C-statistic 0.76 0.69-0.82, adjusted R 2 0.51). Although the addition of PLA2R antibody titer improved the performance of this model (C-statistic: 0.78 0.72-0.84, adjusted R 2 0.61), replacing PLA2R antibody with the combined risk score improved the model further (C-statistic: 0.82 0.77-0.87, adjusted R 2 0.69, difference of C-statistics with clinical model=0.06 0.03-0.10, P < 0.001; difference of C-statistics with clinical-serologic model=0.04 0.01-0.06, P < 0.001).
In patients with PLA2R-associated membranous nephropathy, the combined risk score incorporating inherited risk alleles and PLA2R antibody enhanced the prediction of kidney disease progression compared with PLA2R serology and clinical factors alone.
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