Discovering regulators of ER autophagy using CRISPR screening

Autophagy, the process by which cellular contents are degraded via the lysosome, is critical for cellular homeostasis and regulation. ER autophagy (ER-phagy) is the process by which pieces of the endoplasmic reticulum (ER) are degraded by the lysosome. This process was only recently discovered to occur in mammalian cells, and it has been suggested that misregulation manifests in neuropathy conditions. In order to understand this process, we developed genome-editing tools to aid in uncovering regulators of ER autophagy (ER-phagy).We developed and improved multiple genome-editing tools. In chapter 2, I describe how to use CasRNPs (ribonucleotide proteins) to genome edit cell lines. This protocol is ideal for individual desired edits or to genome-edit in arrayed screening fashion. This chapter details how to make single guide RNAs (sgRNAs), how to purify Cas proteins, and how to use those reagents to edit a cell line.In chapter 3, we used various genome-editing tools (including the protocols described in chapter 2) to uncover regulators of ER autophagy. We conducted a genome-wide screen to identify factors that inhibit or enhance ER-phagy when knocked down. Our screen yielded 200 high-confidence hits. We mechanistically followed up on two pathways: mitochondrial oxidative phosphorylation (OXPHOS) and ER-localized DDRGK1 and UFMylation. This work advances our understanding of the regulatory mechanisms of ER-phagy, and my hope is that the CRISPR tools and ER-phagy tools we generated will allow the ER-phagy field to progress quickly

A Genome-wide ER-phagy Screen Highlights Key Roles of Mitochondrial Metabolism and ER-Resident UFMylation

Selective autophagy of organelles is critical for cellular differentiation, homeostasis, and organismal health. Autophagy of the ER (ER-phagy) is implicated in human neuropathy but is poorly understood beyond a few autophagosomal receptors and remodelers. By using an ER-phagy reporter and genome-wide CRISPRi screening, we identified 200 high-confidence human ER-phagy factors. Two pathways were unexpectedly required for ER-phagy. First, reduced mitochondrial metabolism represses ER-phagy, which is opposite of general autophagy and is independent of AMPK. Second, ER-localized UFMylation is required for ER-phagy to repress the unfolded protein response via IRE1α. The UFL1 ligase is brought to the ER surface by DDRGK1 to UFMylate RPN1 and RPL26 and preferentially targets ER sheets for degradation, analogous to PINK1-Parkin regulation during mitophagy. Our data provide insight into the cellular logic of ER-phagy, reveal parallels between organelle autophagies, and provide an entry point to the relatively unexplored process of degrading the ER network

Chemical Interactions of Polyethylene Glycols (PEGs) and Glycerol with Protein Functional Groups: Applications to Effects of PEG and Glycerol on Protein Processes

In this work, we obtain the data needed to predict chemical interactions of polyethylene glycols (PEGs) and glycerol with proteins and related organic compounds and thereby interpret or predict chemical effects of PEGs on protein processes. To accomplish this, we determine interactions of glycerol and tetraEG with >30 model compounds displaying the major C, N, and O functional groups of proteins. Analysis of these data yields coefficients (α values) that quantify interactions of glycerol, tetraEG, and PEG end (-CH₂OH) and interior (-CH₂OCH₂-) groups with these groups, relative to interactions with water. TetraEG (strongly) and glycerol (weakly) interact favorably with aromatic C, amide N, and cationic N, but unfavorably with amide O, carboxylate O, and salt ions. Strongly unfavorable O and salt anion interactions help make both small and large PEGs effective protein precipitants. Interactions of tetraEG and PEG interior groups with aliphatic C are quite favorable, while interactions of glycerol and PEG end groups with aliphatic C are not. Hence, tetraEG and PEG300 favor unfolding of the DNA-binding domain of lac repressor (lacDBD), while glycerol and di- and monoethylene glycol are stabilizers. Favorable interactions with aromatic and aliphatic C explain why PEG400 greatly increases the solubility of aromatic hydrocarbons and steroids. PEG400–steroid interactions are unusually favorable, presumably because of simultaneous interactions of multiple PEG interior groups with the fused ring system of the steroid. Using α values reported here, chemical contributions to PEG <i>m</i>-values can be predicted or interpreted in terms of changes in water-accessible surface area (ΔASA) and separated from excluded volume effects

Fluorescence Resonance Energy Transfer Characterization of DNA Wrapping in Closed and Open <i>Escherichia coli</i> RNA Polymerase−λP_R Promoter Complexes

Initial recognition of promoter DNA by RNA polymerase (RNAP) is proposed to trigger a series of conformational changes beginning with bending and wrapping of the 40–50 bp of DNA immediately upstream of the −35 region. Kinetic studies demonstrated that the presence of upstream DNA facilitates bending and entry of the downstream duplex (to +20) into the active site cleft to form an advanced closed complex (CC), prior to melting of ∼13 bp (−11 to +2), including the transcription start site (+1). Atomic force microscopy and footprinting revealed that the stable open complex (OC) is also highly wrapped (−60 to +20). To test the proposed bent-wrapped model of duplex DNA in an advanced RNAP−λP_R CC and compare wrapping in the CC and OC, we use fluorescence resonance energy transfer (FRET) between cyanine dyes at far-upstream (−100) and downstream (+14) positions of promoter DNA. Similarly large intrinsic FRET efficiencies are observed for the CC (0.30 ± 0.07) and the OC (0.32 ± 0.11) for both probe orientations. Fluorescence enhancements at +14 are observed in the single-dye-labeled CC and OC. These results demonstrate that upstream DNA is extensively wrapped and the start site region is bent into the cleft in the advanced CC, reducing the distance between positions −100 and +14 on promoter DNA from >300 to <100 Å. The proximity of upstream DNA to the downstream cleft in the advanced CC is consistent with the proposed mechanism for facilitation of OC formation by upstream DNA