Biology subjects

Smith, I. R.

Publications and source records attributed to Smith, I. R..

7 recordsLinked to original sources

bioRxiv2024

Lysosomal storage disease proteo/lipidomic profiling using nMOST links ferritinophagy with mitochondrial iron deficiencies in cells lacking NPC2

Lysosomal storage diseases (LSDs) comprise [~]50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multi-omic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in NPC1-/- and NPC2-/- mutants, where lysosomes accumulate cholesterol. Autophagic and endocytic cargo delivery failures correlated with elevated lyso-phosphatidylcholine species and multi-lamellar structures visualized by cryo-electron tomography. Loss of mitochondrial cristae, MICOS- complex components, and OXPHOS components rich in iron-sulfur cluster proteins in NPC2-/- cells was largely alleviated when iron was provided through the transferrin system. This study reveals how lysosomal dysfunction affects mitochondrial homeostasis and underscores nMOST as a valuable discovery tool for identifying molecular phenotypes across LSDs.

cell biology↗

bioRxiv2023

Combinatorial selective ER-phagy remodels the ER during neurogenesis

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis: a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodeling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of ER and the role of individual ER-phagy receptors is limited. Here, we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodeling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

cell biology↗

bioRxiv2022

Quantitative mapping of autophagic cargo during nutrient stress reveals YIPF3-YIPF4 as membrane receptors for Golgiphagy

During nutrient stress, macroautophagy is employed to degrade cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodeling the proteome. While the machinery responsible for initiation of macroautophagy is well characterized, our understanding of the extent to which individual proteins, protein complexes and organelles are selected for autophagic degradation, and the underlying targeting mechanisms is limited. Here, we use orthogonal proteomic strategies to provide a global molecular inventory of autophagic cargo during nutrient stress in mammalian cell lines. Through prioritization of autophagic cargo, we identify a heterodimeric pair of membrane-embedded proteins, YIPF3 and YIPF4, as receptors for Golgiphagy. During nutrient stress, YIPF4 is mobilized into ATG8-positive vesicles that traffic to lysosomes as measured via Golgiphagy flux reporters in a process that requires the VPS34 and ULK1-FIP200 arms of the autophagy system. Cells lacking YIPF3 or YIPF4 are selectively defective in elimination of Golgi membrane proteins during nutrient stress. By merging absolute protein abundance with autophagic turnover, we create a global protein census describing how autophagic degradation maps onto protein abundance and subcellular localization. Our results, available via an interactive web tool, reveal that autophagic turnover prioritizes membrane-bound organelles (principally Golgi and ER) for proteome remodeling during nutrient stress. One-Sentence SummaryDuring nutrient stress, macroautophagy uses organelle-phagy receptors to prioritize recycling of Golgi and ER membrane proteins.

cell biology↗

bioRxiv2022

PARK15/FBXO7 is dispensable for PINK1/Parkin-dependent mitophagy in iNeuron and HeLa cell systems

The protein kinase PINK1 and ubiquitin ligase Parkin promote removal of damaged mitochondria via a feed-forward mechanism involving ubiquitin (Ub) phosphorylation (pUb), Parkin activation, and ubiquitylation of mitochondrial outer membrane proteins to support recruitment of mitophagy receptors. The ubiquitin ligase substrate receptor FBXO7/PARK15 is mutated in an early-onset parkinsonian-pyramidal syndrome. Previous studies have proposed a role for FBXO7 in promoting Parkin-dependent mitophagy. Here, we systematically examine the involvement of FBXO7 in depolarization and mtUPR-dependent mitophagy in the well-established HeLa and induced-neurons cell systems. We find that FBXO7-/- cells have no demonstrable defect in: 1) kinetics of pUb accumulation, 2) pUb puncta on mitochondria by super-resolution imaging, 3) recruitment of Parkin and autophagy machinery to damaged mitochondria, 4) mitophagic flux, and 5) mitochondrial clearance as quantified by global proteomics. Moreover, global proteomics of neurogenesis in the absence of FBXO7 reveals no obvious alterations in mitochondria or other organelles. These results argue against a general role for FBXO7 in Parkin-dependent mitophagy and point to the need for additional studies to define how FBXO7 mutations promote parkinsonian-pyramidal syndrome.

cell biology↗

bioRxiv2022

Coisolation of peptide pairs for peptide identification and MS/MS-based quantification

SILAC-based metabolic labeling is a widely adopted proteomics approach that enables quantitative comparisons among a variety of experimental conditions. Despite its quantitative capacity, SILAC experiments analyzed with data dependent acquisition (DDA) do not fully leverage peptide pair information for identification and suffer from undersampling compared to label-free proteomic experiments. Herein, we developed a data dependent acquisition strategy that coisolates and fragments SILAC peptide pairs and uses y-ions for their relative quantification. To facilitate the analysis of this type of data, we adapted the Comet sequence database search engine to make use of SILAC peptide paired fragments and developed a tool to annotate and quantify MS/MS spectra of coisolated SILAC pairs. In an initial feasibility experiment, this peptide pair coisolation approach generally improved expectation scores compared to the traditional DDA approach. Fragment ion quantification performed similarly well to precursor quantification in the MS1 and achieved more quantifications. Lastly, our method enables reliable MS/MS quantification of SILAC proteome mixtures with overlapping isotopic distributions, which are difficult to deconvolute in MS1-based quantification. This study demonstrates the initial feasibility of the coisolation approach. Coupling this approach with intelligent acquisition strategies has the potential to improve SILAC peptide sampling and quantification.

systems biology↗

bioRxiv2022

Proteome-wide identification of amino acid substitutions deleterious for protein function

DNA sequencing has led to the discovery of millions of mutations that change the encoded protein sequences, but the impact of nearly all of these mutations on protein function is unknown. We addressed this scarcity of functional data by developing Miro, a proteomic technology that uses mistranslation to introduce amino acid substitutions and biochemical assays to quantify functional differences of thousands of protein variants by mass spectrometry. We apply this technology to the proteome of yeast to reveal amino acid substitutions that impact protein structure, ligand binding, protein-protein interactions, protein post-translational modifications, and protein thermal stability. Adapting Miro to human cells will provide a means to efficiently accelerate our mechanistic interpretation of genomic mutations to predict disease risk.

genomics↗

bioRxiv2020

Identification of phosphosites that alter protein thermal stability

Proteomics has enabled the cataloguing of 100,000s of protein phosphorylation sites 1, however we lack methods to systematically annotate their function. Phosphorylation has numerous biological functions, yet biochemically all involve changes in protein structure and interactions. These biochemical changes can be recapitulated by measuring the difference in stability between the protein and the phosphoprotein. Building on recent work, we present a method to infer phosphosite functionality by reliably measuring such differences at the proteomic scale.

systems biology↗