Lysosome damage triggers acute formation of ER to lysosomes membrane tethers mediated by the bridge-like lipid transport protein VPS13C.

Based on genetic studies, lysosome dysfunction is thought to play a pathogenetic role in Parkinson's disease (PD). Here we show that VPS13C, a bridge-like lipid transport protein and a PD gene, is a sensor of lysosome stress/damage. Upon lysosome membrane perturbation, VPS13C rapidly relocates from the cytosol to the surface of lysosomes where it tethers their membranes to the ER. This recruitment depends on Rab7 and requires release of a brake, most likely an intramolecular interaction within VPS13C, which hinders access of its VAB domain to lysosome-bound Rab7. While another PD protein, LRRK2, is also recruited to stressed/damaged lysosomes, its recruitment occurs at much later stages and by different mechanisms. Given the putative role of VPS13 proteins in bulk lipid transport, these findings suggest lipid delivery to lysosomes by VPS13C is part of an early response to lysosome damage.

In situ architecture of the lipid transport protein VPS13C at ER-lysosome membrane contacts.

VPS13 is a eukaryotic lipid transport protein localized at membrane contact sites. Previous studies suggested that it may transfer lipids between adjacent bilayers by a bridge-like mechanism. Direct evidence for this hypothesis from a full-length structure and from electron microscopy (EM) studies in situ is still missing, however. Here, we have capitalized on AlphaFold predictions to complement the structural information already available about VPS13 and to generate a full-length model of human VPS13C, the Parkinson's disease-linked VPS13 paralog localized at contacts between the endoplasmic reticulum (ER) and endo/lysosomes. Such a model predicts an ∼30-nm rod with a hydrophobic groove that extends throughout its length. We further investigated whether such a structure can be observed in situ at ER-endo/lysosome contacts. To this aim, we combined genetic approaches with cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) to examine HeLa cells overexpressing this protein (either full length or with an internal truncation) along with VAP, its anchoring binding partner at the ER. Using these methods, we identified rod-like densities that span the space separating the two adjacent membranes and that match the predicted structures of either full-length VPS13C or its shorter truncated mutant, thus providing in situ evidence for a bridge model of VPS13 in lipid transport.

ER-lysosome lipid transfer protein VPS13C/PARK23 prevents aberrant mtDNA-dependent STING signaling.

Mutations in VPS13C cause early-onset, autosomal recessive Parkinson's disease (PD). We have established that VPS13C encodes a lipid transfer protein localized to contact sites between the ER and late endosomes/lysosomes. In the current study, we demonstrate that depleting VPS13C in HeLa cells causes an accumulation of lysosomes with an altered lipid profile, including an accumulation of di-22:6-BMP, a biomarker of the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. In addition, the DNA-sensing cGAS-STING pathway, which was recently implicated in PD pathogenesis, is activated in these cells. This activation results from a combination of elevated mitochondrial DNA in the cytosol and a defect in the degradation of activated STING, a lysosome-dependent process. These results suggest a link between ER-lysosome lipid transfer and innate immune activation in a model human cell line and place VPS13C in pathways relevant to PD pathogenesis.

Assessing HDL Metabolism in Subjects with Elevated Levels of HDL Cholesterol and Coronary Artery Disease.

High-density lipoprotein cholesterol (HDL-C) is thought to be atheroprotective yet some patients with elevated HDL-C levels develop cardiovascular disease, possibly due to the presence of dysfunctional HDL. We aimed to assess the metabolic fate of circulating HDL particles in patients with high HDL-C with and without coronary artery disease (CAD) using in vivo dual labeling of its cholesterol and protein moieties. We measured HDL apolipoprotein (apo) A-I, apoA-II, free cholesterol (FC), and cholesteryl ester (CE) kinetics using stable isotope-labeled tracers (D3-leucine and 13C2-acetate) as well as ex vivo cholesterol efflux to HDL in subjects with (n = 6) and without (n = 6) CAD that had HDL-C levels >90th percentile. Healthy controls with HDL-C within the normal range (n = 6) who underwent the same procedures were used as the reference. Subjects with high HDL-C with and without CAD had similar plasma lipid levels and similar apoA-I, apoA-II, HDL FC, and CE pool sizes with no significant differences in fractional clearance rates (FCRs) or production rates (PRs) of these components between groups. Subjects with high HDL-C with and without CAD also had similar basal and cAMP-stimulated ex vivo cholesterol efflux to HDL. When all subjects were considered (n = 18), unstimulated non-ABCA1-mediated efflux (but not ABCA1-specific efflux) was correlated positively with apoA-I production (r = 0.552, p = 0.017) and HDL FC and CE pool sizes, and negatively with the fractional clearance rate of FC (r = -0.759, p = 4.1 × 10-4) and CE (r = -0.652, p = 4.57 × 10-3). Our data are consistent with the concept that ex vivo non-ABCA1 efflux capacity may correlate with slower in vivo turnover of HDL cholesterol moieties. The use of a dual labeling protocol provided for the first time the opportunity to assess the association of ex vivo cholesterol efflux capacity with in vivo HDL cholesterol metabolic parameters.

Role of VPS13, a protein with similarity to ATG2, in physiology and disease.

The evolutionarily conserved VPS13 family proteins have been implicated in several cellular processes. Mutations in each of the four human VPS13s cause neurodevelopmental or neurodegenerative disorders. Until recently, the molecular function of VPS13 remained elusive. Genetic, functional and structural studies have now revealed that VPS13 acts at contact sites between intracellular organelles to transport lipids by a novel mechanism: direct transfer between bilayers via a hydrophobic channel that spans its entire rod-like N-terminal half. Predicted similarities to the autophagy protein ATG2 suggested a similar role for ATG2 that has now been confirmed by structural and functional studies. Here, after a brief review of this evidence, we discuss what is known of human VPS13 proteins in physiology and disease.

VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites.

Mutations in the human VPS13 genes are responsible for neurodevelopmental and neurodegenerative disorders including chorea acanthocytosis (VPS13A) and Parkinson's disease (VPS13C). The mechanisms of these diseases are unknown. Genetic studies in yeast hinted that Vps13 may have a role in lipid exchange between organelles. In this study, we show that the N-terminal portion of VPS13 is tubular, with a hydrophobic cavity that can solubilize and transport glycerolipids between membranes. We also show that human VPS13A and VPS13C bind to the ER, tethering it to mitochondria (VPS13A), to late endosome/lysosomes (VPS13C), and to lipid droplets (both VPS13A and VPS13C). These findings identify VPS13 as a lipid transporter between the ER and other organelles, implicating defects in membrane lipid homeostasis in neurological disorders resulting from their mutations. Sequence and secondary structure similarity between the N-terminal portions of Vps13 and other proteins such as the autophagy protein ATG2 suggest lipid transport roles for these proteins as well.

Multiplexed Targeted Resequencing Identifies Coding and Regulatory Variation Underlying Phenotypic Extremes of High-Density Lipoprotein Cholesterol in Humans.

BACKGROUND: Genome-wide association studies have uncovered common variants at many loci influencing human complex traits, such as high-density lipoprotein cholesterol (HDL-C). However, the contribution of the identified genes is difficult to ascertain from current efforts interrogating common variants with small effects. Thus, there is a pressing need for scalable, cost-effective strategies for uncovering causal variants, many of which may be rare and noncoding. METHODS: Here, we used a molecular inversion probe target capture approach to resequence both coding and regulatory regions at 7 HDL-C-associated loci in 797 individuals with extremely high HDL-C versus 735 low-to-normal HDL-C controls. Our targets included protein-coding regions of GALNT2, APOA5, APOC3, SCARB1, CCDC92, ZNF664, CETP, and LIPG (>9 kb) and proximate noncoding regulatory features (>42 kb). RESULTS: Exome-wide genotyping in 1114 of the 1532 participants yielded a >90% genotyping concordance rate with molecular inversion probe-identified variants in ≈90% of participants. This approach rediscovered nearly all established genome-wide association studies associations in GALNT2, CETP, and LIPG loci with significant and concordant associations with HDL-C from our phenotypic extremes design at 0.1% of the sample size of lipid genome-wide association studies. In addition, we identified a novel, rare, CETP noncoding variant enriched in the extreme high HDL-C group (P<0.01, score test). CONCLUSIONS: Our targeted resequencing of individuals at the HDL-C phenotypic extremes offers a novel, efficient, and cost-effective approach for identifying rare coding and noncoding variation differences in extreme phenotypes and supports the rationale for applying this methodology to uncover rare variation-particularly noncoding variation-underlying myriad complex traits.

Paradoxical coronary artery disease in humans with hyperalphalipoproteinemia is associated with distinct differences in the high-density lipoprotein phosphosphingolipidome.

BACKGROUND: Plasma high-density lipoprotein cholesterol (HDL-C) levels are inversely associated with risk of coronary artery disease (CAD) in epidemiologic studies. Despite this, the directionality of this relationship and the underlying biology behind it remain to be firmly established, especially at the extremes of HDL-C levels. OBJECTIVE: We investigated differences in the HDL phosphosphingolipidome in a rare population of subjects with premature CAD despite high HDL-C levels to gain insight into the association between the HDL lipidome and CAD disease status in this unusual phenotype. We sought to assess differences in HDL composition that are associated with CAD in subjects with HDL-C >90th percentile. We predicted that quantitative lipidomic analysis of HDL particles would reveal novel differences between CAD patients and healthy subjects with matched HDL-C levels. METHODS: We collected plasma samples from 25 subjects with HDL-C >90th percentile and clinically manifest CAD and healthy controls with HDL-C >90th percentile and without self-reported CAD. More than 140 individual HDL phospholipid and sphingolipid species were analyzed by LC/MS/MS. RESULTS: Significant reductions in HDL phosphatidylcholine (-2.41%, Q value = 0.025) and phosphatidylinositol (-10.7%, Q value = 0.047) content, as well as elevated sphingomyelin (+10.0%, Q value = 0.025) content, and sphingomyelin/phosphatidylcholine ratio (+12.8%, P value = .005) were associated with CAD status in subjects with high HDL-C. CONCLUSIONS: These differences may lay the groundwork for further analysis of the relationship between the HDL lipidome and disease states, as well as for the development of biomarkers of CAD status and HDL function.

Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease.

Scavenger receptor BI (SR-BI) is the major receptor for high-density lipoprotein (HDL) cholesterol (HDL-C). In humans, high amounts of HDL-C in plasma are associated with a lower risk of coronary heart disease (CHD). Mice that have depleted Scarb1 (SR-BI knockout mice) have markedly elevated HDL-C levels but, paradoxically, increased atherosclerosis. The impact of SR-BI on HDL metabolism and CHD risk in humans remains unclear. Through targeted sequencing of coding regions of lipid-modifying genes in 328 individuals with extremely high plasma HDL-C levels, we identified a homozygote for a loss-of-function variant, in which leucine replaces proline 376 (P376L), in SCARB1, the gene encoding SR-BI. The P376L variant impairs posttranslational processing of SR-BI and abrogates selective HDL cholesterol uptake in transfected cells, in hepatocyte-like cells derived from induced pluripotent stem cells from the homozygous subject, and in mice. Large population-based studies revealed that subjects who are heterozygous carriers of the P376L variant have significantly increased levels of plasma HDL-C. P376L carriers have a profound HDL-related phenotype and an increased risk of CHD (odds ratio = 1.79, which is statistically significant).

Gas phase thermal reactions of exo-8-cyclopropyl-bicyclo[4.2.0]oct-2-ene (1-exo).

The title compound 1-exo (with minor amounts of its C8 epimer 1-endo) was prepared by Wolff-Kishner reduction of the cycloadduct of 1,3-cyclohexadiene and cyclopropylketene. The [1,3]-migration product 2-endo was synthesized by efficient selective cyclopropanation of endo-5-vinylbicyclo[2.2.2]oct-2-ene at the exocyclic π-bond. Gas phase thermal reactions of 1-exo afforded C8 epimerization to 1-endo, [1,3]- migrations to 2-exo and 2-endo, direct fragmentation to cyclohexadiene and vinylcyclopropane, and CPC rearrangement in the following relative kinetic order: kep > k13 > kf > kCPC.

Thermal isomerizations of cis,anti,cis-tricyclo[7.4.0.0(2,8)]tridec-10-ene.

cis,anti,cis-Tricyclo[7.4.0.0(2,8)]tridec-10-ene (13TCT) undergoes [1,3] sigmatropic rearrangements at 315 °C in the gas phase to the si product 1 and to the sr product 2 with si/sr = 2.1. The dominant thermal isomerization process, however, is epimerization at C8 to afford product 3. That stereomutation at C8 occurs 50% faster than the si and sr shifts combined.