Biology subjects

Hancock-Cerutti, W. F.

Publications and source records attributed to Hancock-Cerutti, W. F..

3 recordsLinked to original sources

bioRxiv2022

In situ architecture of the lipid transport protein VPS13C at ER-lysosomes 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 EM studies in situ, however, is still missing. 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 Parkinsons disease-linked VPS13 paralog localized at contacts between the ER and endo/lysosomes. Such model predicts a ~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 the first in-situ evidence for a bridge-model of VPS13 in lipid transport. Intriguingly, the majority of the VPS13C rods were separated from the ER membranes by a narrow gap, suggesting that while VAP anchors the protein to the ER, direct contact of the VPS13C rod with the ER bilayer to allow lipid transport may be independently regulated.

cell biology↗

bioRxiv2021

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

Mutations in VPS13C cause early onset, autosomal recessive Parkinsons Disease (PD). We have established that VPS13C encodes a lipid transfer protein localized to contact sites between the endoplasmic reticulum (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 and place VPS13C in pathways relevant to PD pathogenesis.

cell biology↗

bioRxiv2017

Multiplexed targeted resequencing identifies coding and regulatory variation underlying phenotypic extremes of HDL-cholesterol in humans

Genome-wide association studies have uncovered common variants at many loci influencing human complex traits and diseases, 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. Here, we used a multiplexed inversion probe (MIP) target capture approach to resequence both coding and regulatory regions at seven HDL-C associated loci in 797 individuals with extremely high HDL-C vs. 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). Exome-wide genotyping in 1,114 of the 1,532 participants yielded a >90% genotyping concordance rate with MIP-identified variants in ~90% of participants. This approach rediscovered nearly all established GWAS 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 GWAS studies. In addition, we identified a novel, rare, CETP noncoding variant enriched in the extreme high HDL-C group (P<0.01, Score Test). 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 non-coding variation--underlying myriad complex traits.

genomics↗