Biology subjects

Zoncu, R.

Publications and source records attributed to Zoncu, R..

9 recordsLinked to original sources

bioRxiv2025

PCSK9 dependent cholesterol acquisition and utilization underlies metastatic organ preference in pancreatic cancer.

To grow at distant sites, metastatic cells must overcome major challenges posed by the unique cellular and metabolic composition of secondary organs1. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that metastasizes to the liver and lungs. Despite evidence of metabolic reprogramming away from the primary site, the key drivers that dictate the ability of PDAC cells to colonize the liver or lungs and survive there are undefined. We identified Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) as predictive of colonization of the liver versus lungs by integrating datasets describing the metastatic tropism of human PDAC cell lines2, with in vivo metastasis modeling in mice and gene expression correlation analysis. PCSK9 is a negative regulator of low density lipoprotein(LDL)-cholesterol import and, accordingly, PCSK9-low PDAC cells exhibit strong preference for colonizing LDL-rich liver tissue. Cholesterol taken up by liveravid PCSK9-low cells is converted into the signaling oxysterol, 24(S)-hydroxycholesterol, which reprograms the surrounding microenvironment to induce nutrient release from neighboring hepatocytes. Conversely, PCSK9-high, lung-avid PDAC cells rely on transcriptional upregulation of the distal cholesterol synthesis pathway to generate intermediates, chiefly, 7dehydrodesmosterol and 7-dehydrocholesterol, with protective action against ferroptosis, a vulnerability in the oxygen-rich microenvironment of the lung. Increasing PCSK9 levels redirected liver-avid cells to the lung whereas its ablation drove lung-avid cells to the liver, thereby establishing PCSK9 as necessary and sufficient for secondary organ site preference. Our studies reveal PCSK9-driven differential utilization of the distal cholesterol synthesis pathway as a key and potentially actionable driver of metastatic growth in PDAC.

cancer biology↗

bioRxiv2024

Leucine Aminopeptidase LyLAP enables lysosomal degradation of membrane proteins

Proteolysis of hydrophobic helices is required for complete breakdown of every transmembrane protein trafficked to the lysosome and sustains high rates of endocytosis. However, the lysosomal mechanisms for degrading hydrophobic domains remain unknown. Combining lysosomal proteomics with functional genomic data mining, we identify Lysosomal Leucine Aminopeptidase (LyLAP; formerly Phospholipase B Domain-Containing 1) as the hydrolase most tightly associated with elevated endocytic activity. Untargeted metabolomics and biochemical reconstitution demonstrate that LyLAP is not a phospholipase, but a processive monoaminopeptidase with strong preference for N-terminal leucine - an activity necessary and sufficient for breakdown of hydrophobic transmembrane domains. LyLAP is upregulated in pancreatic ductal adenocarcinoma (PDA), which relies on macropinocytosis for nutrient uptake, and its ablation led to buildup of undigested hydrophobic peptides, which compromised lysosomal membrane integrity and inhibited PDA cell growth. Thus, LyLAP enables lysosomal degradation of membrane proteins, and may represent a vulnerability in highly endocytic cancer cells. One sentence summaryLyLAP degrades transmembrane proteins to sustain high endocytosis and lysosomal membrane stability in pancreatic cancer.

biochemistry↗

bioRxiv2024

mTORC1 Signaling Inhibition Modulates Mitochondrial Function in Frataxin Deficiency

Lysosomes regulate mitochondrial function through multiple mechanisms including the master regulator, mechanistic Target of Rapamycin Complex 1 (mTORC1) protein kinase, which is activated at the lysosomal membrane by nutrient, growth factor and energy signals. mTORC1 promotes mitochondrial protein composition changes, respiratory capacity, and dynamics, though the full range of mitochondrial-regulating functions of this protein kinase remain undetermined. We find that acute chemical modulation of mTORC1 signaling decreased mitochondrial oxygen consumption, increased mitochondrial membrane potential and reduced susceptibility to stress-induced mitophagy. In cellular models of Friedreichs Ataxia (FA), where loss of the Frataxin (FXN) protein suppresses Fe-S cluster synthesis and mitochondrial respiration, the changes induced by mTORC1 inhibitors lead to improved cell survival. Proteomic-based profiling uncover compositional changes that could underlie mTORC1-dependent modulation of FXN-deficient mitochondria. These studies highlight mTORC1 signaling as a regulator of mitochondrial composition and function, prompting further evaluation of this pathway in the context of mitochondrial disease.

cell biology↗

bioRxiv2024

Autophagosomes coordinate an AKAP11-dependent regulatory checkpoint that shapes neuronal PKA signaling

Protein Kinase A (PKA) is regulated spatially and temporally via scaffolding of its catalytic (C/{beta}) and regulatory (RI/RII) subunits by the A-kinase-anchoring proteins (AKAP). PKA engages in poorly understood interactions with autophagy, a key degradation pathway for neuronal cell homeostasis, partly via its AKAP11 scaffold. Mutations in AKAP11 drive schizophrenia and bipolar disorders (SZ-BP) through unknown mechanisms. Through proteomic-based analysis of immunopurified lysosomes, we identify the C-RI-AKAP11 holocomplex as a prominent autophagy-associated protein kinase complex. AKAP11 scaffolds C-RI to the autophagic machinery via its LC3-interacting region (LIR), enabling both PKA regulation by upstream signals, and its autophagy-dependent degradation. We identify Ser83 on the RI linker-hinge region as an AKAP11-dependent phospho-residue that modulates RI-C binding and cAMP-induced PKA activation. Decoupling AKAP11-PKA from autophagy alters Ser83 phosphorylation, supporting an autophagy-dependent checkpoint for PKA signaling. Ablating AKAP11 in induced pluripotent stem cell-derived neurons reveals dysregulation of multiple pathways for neuronal homeostasis. Thus, the autophagosome is a novel platform that modulate PKA signaling, providing a possible mechanistic link to SZ/BP pathophysiology.

cell biology↗

bioRxiv2022

Structural basis for FLCN RagC GAP activation in MiT-TFE substrate-selective mTORC1 regulation

mTORC1 regulates cell growth and catabolism in response to fluctuations in nutrients through phosphorylation of key substrates. The tumor suppressor FLCN is a RagC GTPase activating protein (GAP) that regulates mTORC1 phosphorylation of TFEB, controlling lysosome biogenesis and autophagy. Here, we determined the cryo-EM structure of the active FLCN complex (AFC) containing FLCN, FNIP2, the N-terminal tail of SLC38A9, the RagAGDP:RagCGDP.BeFx- GTPase dimer, and the Ragulator scaffold. Relative to the inactive lysosomal FLCN complex (LFC) structure, FLCN reorients by 90{degrees}, breaks its contacts with RagA, and makes new contacts with RagC that position its Arg164 finger for catalysis. Disruption of the AFC-specific interfaces of FLCN and FNIP2 with RagC eliminated GAP activity in vitro and led to nuclear retention of TFE3, with no effect on mTORC1 phosphorylation of S6K or 4E-BP1. The structure thus provides a roadmap to discover TFEB-selective mTORC1 antagonists. One-Sentence SummaryThe cryo-EM structure of the active FLCN RagC GAP complex provides a structural basis for TFEB/TFE3 substrate-selective targeting of mTORC1.

biochemistry↗

bioRxiv2021

Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth

Lysosomes must maintain integrity of their limiting membrane to ensure efficient fusion with incoming organelles and degradation of substrates within their lumen. Pancreatic cancer cells upregulate lysosomal biogenesis to enhance nutrient recycling and stress resistance, but whether dedicated programs for maintaining lysosomal membrane integrity facilitate pancreatic cancer growth is unknown. Using proteomic-based organelle profiling, we identify the Ferlin family plasma membrane repair factor, Myoferlin, as selectively and highly enriched on the membrane of pancreatic cancer lysosomes. Mechanistically, lysosome localization of Myoferlin is necessary and sufficient for maintenance of lysosome health and provides an early-acting protective system against membrane damage that is independent from the endosomal sorting complex required for transport (ESCRT)-mediated repair network. Myoferlin is upregulated in human pancreatic cancer, predicts poor survival, and its ablation severely impairs lysosome function and tumour growth in vivo. Thus, retargeting of plasma membrane repair factors enhances pro-oncogenic activities of the lysosome.

cancer biology↗

bioRxiv2020

4 β-hydroxycholesterol is a pro-lipogenic factor that promotes SREBP1c expression and activity through Liver X-receptor.

Oxysterols are oxidized derivatives of cholesterol that play signaling roles in lipid biosynthesis and homeostasis. Here we show that 4{beta}-hydroxycholesterol (4{beta}-HC), a liver and serum abundant oxysterol of poorly defined function, is a potent and selective inducer of the master lipogenic transcription factor, Sterol Regulatory Element Binding Protein 1c (SREBP1c), but not the related steroidogenic transcription factor SREBP2. Mechanistically, 4{beta}-HC acts as a putative agonist for Liver X receptor (LXR), a sterol sensor and transcriptional regulator previously linked to SREBP1c activation. Unique among the oxysterol agonists of LXR, 4{beta}-HC induced expression of the lipogenic program downstream of SREBP1c, and triggered de novo lipogenesis both in primary hepatocytes and in mouse liver. 4{beta}-HC-acted in parallel to insulin-PI3K-dependent signaling to stimulate triglyceride synthesis and lipid droplet accumulation. Thus, 4{beta}-HC is an endogenous regulator of de novo lipogenesis through the LXR-SREBP1c axis.

biochemistry↗

bioRxiv2020

NPC1-mTORC1 signaling Couples Cholesterol Sensing to Organelle Homeostasis and is a Targetable Pathway in Niemann-Pick type C

Lysosomes promote cellular homeostasis through macromolecular hydrolysis within their lumen and metabolic signaling by the mTORC1 kinase on their limiting membranes. Both hydrolytic and signaling functions require precise regulation of lysosomal cholesterol content. In Niemann-Pick type C (NPC), loss of the cholesterol exporter, NPC1, causes cholesterol accumulation within lysosomes, leading to mTORC1 hyperactivation, disrupted mitochondrial function and neurodegeneration. The compositional and functional alterations in NPC lysosomes, and how aberrant cholesterol-mTORC1 signaling contributes to organelle pathogenesis are not understood. Through proteomic profiling of NPC lysosomes, we find pronounced proteolytic impairment compounded with hydrolase depletion and enhanced membrane damage. Genetic and pharmacologic mTORC1 inhibition restores lysosomal proteolysis without correcting cholesterol storage, implicating aberrant mTORC1 as a pathogenic driver downstream of cholesterol accumulation. Consistently, mTORC1 inhibition ameliorates mitochondrial dysfunction in a neuronal model of NPC. Thus, cholesterol-mTORC1 signaling controls organelle homeostasis and is a targetable pathway in NPC.

cell biology↗

bioRxiv2020

Structure of the lysosomal SCARF (L-SCARF) complex, an Arf GAP haploinsufficient in ALS and FTD

Mutation of C9ORF72 is the most prevalent defect in amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD). Together with hexanucleotide repeat expansion, haploinsufficiency of C9ORF72 contributes to neuronal dysfunction. We determined the structure of the SMCR8-C9orf72-WDR41 complex by cryo-EM. C9orf72 and SMCR8 are both longin-DENN domain proteins, while WDR41 is a beta-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and SMCR8DENN drive lysosomal localization in amino acid starvation. The structure suggested that SMCR8-C9orf72 was a small GTPase activating protein (GAP). We found that SMCR8-C9orf72-WDR41 is a GAP for Arf family small GTPases, and refer to it as the Lysosomal SMCR8-C9orf72 Arf GAP ("L-SCARF") complex. These data rationalize the function of C9orf72 both in normal physiology and in ALS/FTD.

biochemistry↗