Noam Zelcer

Neutral glucosylceramidase GBA2 is a retaining β-d-glucosidase involved in sphingolipid metabolism and implicated in neurological lysosomal storage disorders, yet the lack of highly selective chemical inhibitors has hindered efforts to disentangle its function from that of glucosylceramide synthase (GCS) and lysosomal glucosylceramidase (GBA1). Here, we report the development of the first brain-penetrant, covalent inhibitor that selectively inactivates GBA2 in vivo. Building on β-d-arabinofuranose-configured (β-d-Araf) cyclitol aziridine activity-based probes previously shown to covalently and selectively label GBA2, we designed and synthesized a focused library of electrophile-bearing cyclitols, including epoxides, cyclic sulfates, and a diverse set of N-alkyl aziridines. Biochemical evaluation of their inhibitory activity against GBA1, GBA2, GBA3 and GCS revealed that several N-alkyl aziridines are highly selective, nanomolar GBA2 inhibitors. Lead compounds also demonstrated metabolic stability in murine plasma, robust cellular activity and high selectivity in living cells, with no detectable GCS inhibition. Notably, octyl-alkyne aziridine 5 selectively inhibited GBA2 in mouse brain, representing the first covalent GBA2 inhibitor with demonstrated in vivo activity and brain permeability. Together, these results establish β-d-Araf cyclitol aziridine 5 as a versatile inhibitor for selective, covalent GBA2 inhibition, well suited to dissect GBA2 biology in vivo, particularly in neurological lysosomal storage disorders.

Human CD4 ⁺ T cells utilize nutrients, including lipids, to support their activation and polarization. Considering the pivotal role of lipoproteins in lipid transport, we reasoned that lipoprotein uptake and processing could effect CD4 ⁺ T cell function. Here, we demonstrate that activation of human CD4 ⁺ T cells induced expression of LDL receptor (LDLR) to facilitate LDLR-mediated endocytosis of LDL. Degradation of surface LDLR on CD4 ⁺ T cells with PCSK9 hampered activation and proliferation of the cells. Lipoprotein deprivation or blocking of lysosomal cholesterol egress impaired activation of mechanistic target of rapamycin complex 1 (mTORC1), affecting CD4 ⁺ T cell activation and proliferation. Furthermore, lipoprotein deprivation of cultured primary CD4 ⁺ T cells lead to reduced expression of c-MAF and FOXP3, key transcription factors for IL-10, accompanied by reduced IL-10 secretion. The pivotal role of LDLR-mediated lipoprotein uptake for mTORC1 activity, c-MAF and FOXP3 expression, and IL-10 secretion was confirmed using LDLR-dysfunctional CD4 ⁺ T cells from patients with homozygous familial hypercholesterolemia. Our study offers valuable insights into the lipoprotein metabolism of human CD4 ⁺ T cells and their reliance on the LDLR pathway for activation and polarization, a feature that may be leveraged to modulate CD4 ⁺ T cell function.

Charcot–Marie–Tooth Disease Type 2P (CMT2P) is an inherited axonal neuropathy caused by dominant mutations in the RING domain of LRSAM1. The precise cellular consequences of these mutations remain unclear. Here, we investigated the subcellular localization of wild‐type and RING mutant LRSAM1 in multiple cell models, including patient‐derived fibroblasts and neuronal cell lines. Using immunocytochemistry, membrane and lipid droplet fractionation, and colocalization with organelle markers, we found that RING mutant LRSAM1 consistently mislocalized, deviating from its typical diffuse cytosolic pattern to form large perinuclear aggregates. This mislocalization also caused abnormal clustering of the LRSAM1 target protein TSG101 that coaggregated with mutant LRSAM1. Wild‐type LRSAM1 predominantly localized to the cytosolic fraction, whereas the mutant protein shifted toward the membranous fraction. Wild‐type LRSAM1 was also observed in the lipid droplet fraction, whereas mutant LRSAM1 was absent. Our findings demonstrate that pathogenic RING mutations in LRSAM1 disrupt its subcellular localization and that of its targets. These results highlight protein mislocalization as a pathogenic mechanism in CMT2P, providing new insight into disease etiology.

The Niemann-Pick type 1 and 2 proteins (NPC1 and NPC2) coordinate cholesterol egress from late endosomes-lysosomes (LE/LY). Proper folding, trafficking, and localization of both NPC proteins are essential for normal LE/LY cholesterol handling. Accordingly, mutations in NPC genes cause Niemann–Pick type C (NPC) disease, a progressive neurodegenerative lysosomal cholesterol storage disorder. The routes by which NPC1 reaches the LE/LY compartment in mammalian cells are not fully elucidated. Therefore, to interrogate NPC1 trafficking, we developed genome-engineered HeLa cells expressing endogenous NPC1 ^mNeon . We demonstrate that endogenous NPC1 localizes to the LE/LY compartment and by using protein proximity-based approaches that NPC1 resides in the same membranes as Vacuolar Protein Sorting-associated protein 41 (VPS41), one of the two unique subunits of the homotypic fusion and vacuole protein sorting complex. Loss of VPS41 increases NPC1 and Lysosomal Associated Membrane Protein 1 (LAMP1) abundance. Paradoxically, this results in marked accumulation of lysosomal cholesterol and induction of sterol regulatory element-binding protein signaling. Mechanistically, using immuno-fluorescence and electron microscopy imaging in combination with a VPS41-dependent ectopic recruitment assay, we demonstrate that this is due to a shift in the localization of NPC1 and LAMP1 from LE/LY to biosynthetic vesicles called LAMP carriers. These vesicles have been recently described to transport lysosomal-destined cargo directly from the trans- Golgi (TGN) network to LE/LY. In conclusion, we identify NPC1 as a cargo for VPS41-dependent LAMP carriers that are instrumental for the delivery of NPC1 to LE/LY and maintaining cellular cholesterol homeostasis.

The chimeric cytokine IC7Fc conveys the metabolic signaling properties of the glycoprotein 130 receptor cytokines interleukin-6 and ciliary neurotrophic factor via membrane-bound signaling. IC7Fc was previously shown to slow the progression of type 2 diabetes mellitus, and here, we demonstrate its effect on atherosclerotic development. In APOE*3-Leiden.CETP mice, an atherosclerosis-prone model with a humanized lipoprotein metabolism, IC7Fc markedly lowered plasma triglyceride and total cholesterol levels. This was mechanistically explained by an inhibition of de novo lipogenesis in the liver, increased synthesis of bile acids from cholesterol, and down-regulated apolipoprotein B synthesis, which resulted in decreased cholesterol secretion in very low–density lipoprotein particles. As a consequence, IC7Fc treatment considerably reduced atherosclerotic lesion formation and vascular inflammation compared with current antihyperlipidemic therapy. In conclusion, IC7Fc is a promising pharmacological treatment for cardiometabolic diseases targeting hyperlipidemia and inflammation.

The accumulation of foamy macrophages is a pathological hallmark of demyelinating brain disorders. Perturbed metabolism and efflux of intracellular lipids underlie the development of a harmful foamy macrophage phenotype in these disorders, yet, the molecular mechanisms underlying this dysregulation are poorly understood. Here, we show that the ubiquitin-proteasome system controls the turnover of the cholesterol efflux transporter ATP-binding cassette A1 (ABCA1) in lipid-loaded macrophages in the brain. We report that accumulation of myelin-derived lipids promotes the abundance and activity of ubiquitin-protein E3 ligase A (UBE3A) in macrophages, which stimulates ABCA1 ubiquitination and subsequent degradation. This boosts cellular lipid accumulation and induces an inflammatory macrophage phenotype that impairs remyelination. We further establish Tat-interacting protein 30 (TIP30), an inhibitor of importin β-mediated nuclear import, as an essential regulator of cytosolic UBE3A levels. Together, our findings identify UBE3A as a driver of foam cell formation and indicate that targeting UBE3A-mediated ABCA1 degradation is a promising strategy to enhance central nervous system repair.

Purpose of review: Lipid metabolism and de-novo lipogenesis (DNL) is broadly controlled by the SREBP transcription factors. These transcription factors are matured from membrane-anchored precursor proteins by the proteolytic actions of the proteases S1P and S2P. In this review, we summarize the current understanding of SPRING, a recently identified activator of S1P. Recent findings: Recent studies of SPRING using animal, cellular, biochemical, and biophysical methods have established SPRING as a core component of the SREBP machinery. Deletion of SPRING in cells and animal livers specifically reduces SREBP activity yet leaves other S1P substrates intact, demonstrating an SREBP-specific role for SPRING in licensing S1P activity. Mechanistic biochemical and structural studies revealed that SPRING activates S1P by competitively displacing its inhibitory pro-domain and elucidated how small molecule inhibition of S1P can be accomplished. Summary: Current studies have shown how SPRING activates S1P and uncovered a critical role for SPRING in the SREBP pathway. Further studies are warranted to understand this emerging, connection between SPRING and the regulation of DNL through SREBP.

Site-one protease (S1P) carries out the first proteolytic step to activate membrane-bound effector proteins in the Golgi. S1P matures through an autocatalytic process that begins in the endoplasmic reticulum (ER) and culminates with the displacement of its inhibitory pro-domain by its cofactor, sterol regulatory element binding protein-regulating gene (SPRING). Spatial control of S1P activity and substrate localization underpins signaling pathways governing, among others, lipogenesis, ER stress, and lysosome biogenesis. The factors governing these pathways are activated by S1P-mediated proteolysis upon their regulated transport from the ER to the Golgi. S1P cleaves substrates with the recognition sequence RX(L/I/V)Z, where X is any residue other than Cys or Pro and Z is preferably Leu or Lys. However, the structural basis for substrate recognition by S1P has remained unknown. Here, we used the small molecule PF-429242, a competitive inhibitor of S1P, to investigate substrate recognition by the S1P/SPRING complex. We determined the structure of S1P/SPRING bound to PF-429242 and found that PF-429242 binds S1P in the same pocket that recognizes the substrate’s conserved P ₄ Arg. Further structural analysis suggests that S1P requires a conformation change to accommodate the substrate’s P ₂ (L/I/V) residue. We designed an S1P mutation (I308A) to reduce the steric clash at the P ₂ position and generated an S1P that was resistant to PF-429242 in biochemical and cell culture assays. Our findings reveal selectivity in the recognition of substrates by S1P and provide a roadmap for the rational design of improved S1P inhibitors.