Protein Synthesis and Degradation
Ectopic RING activity at the ER membrane differentially impacts ERAD protein quality control pathways
Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway that ensures misfolded proteins are removed from the ER and destroyed. In ERAD, membrane and luminal substrates are ubiquitylated by ER-resident RING-type E3 ubiquitin ligases, retrotranslocated into the cytosol, and degraded by the proteasome. Overexpression of ERAD factors is frequently used in yeast and mammalian cells to study this process. Here, we analyze the impact of ERAD E3 overexpression on substrate turnover in yeast, where there are three ERAD E3 complexes (Doa10, Hrd1, and Asi1-3).E3 ligase adaptor FBXO7 contributes to ubiquitination and proteasomal degradation of SIRT7 and promotes cell death in response to hydrogen peroxide
Parkinson’s disease (PD) is a degenerative disorder of the central nervous system that affects 1% of the population over the age of 60. Although aging is one of the main risk factors for PD, the pathogenic mechanism of this disease remains unclear. Mutations in the F-box-only protein 7 (FBXO7) gene have been previously found to cause early onset autosomal recessive familial PD. FBXO7 is an adaptor protein in the SKP1–Cullin–1–F-box (SCF) E3 ligase complex that facilitates the ubiquitination of substrates.Assembly chaperone Nas6 selectively destabilizes 26S proteasomes with defective regulatory particle-core particle interfaces
The 26S proteasome is a 66-subunit–chambered protease present in all eukaryotes that maintains organismal health by degrading unneeded or defective proteins. Defects in proteasome function or assembly are known to contribute to the development of various cancers, neurodegeneration, and diabetes. During proteasome biogenesis, a family of evolutionarily conserved chaperones assembles a hexameric ring of AAA+ family ATPase subunits contained within the proteasomal regulatory particle (RP) and guide their docking onto the surface of the proteolytic core particle (CP).RanBP9 controls the oligomeric state of CTLH complex assemblies
The CTLH (C-terminal to lissencephaly-1 homology motif) complex is a multisubunit RING E3 ligase with poorly defined substrate specificity and flexible subunit composition. Two key subunits, muskelin and Wdr26, specify two alternative CTLH complexes that differ in quaternary structure, thereby allowing the E3 ligase to presumably target different substrates. With the aid of different biophysical and biochemical techniques, we characterized CTLH complex assembly pathways, focusing not only on Wdr26 and muskelin but also on RanBP9, Twa1, and Armc8β subunits, which are critical to establish the scaffold of this E3 ligase.The penultimate step of proteasomal ATPase assembly is mediated by a switch dependent on the chaperone Nas2
The proteasome holoenzyme is a complex molecular machine that degrades most proteins. In the proteasome holoenzyme, six distinct ATPase subunits (Rpt1 through Rpt6) enable protein degradation by injecting protein substrates into it. Individual Rpt subunits assemble into a heterohexameric “Rpt ring” in a stepwise manner, by binding to their cognate chaperones. Completion of the heterohexameric Rpt ring correlates with release of a specific chaperone, Nas2; however, it is unclear whether and how this event may ensure proper Rpt ring assembly.An RNA stem-loop functions in conjunction with an upstream open reading frame to direct preferential translation in the integrated stress response
In response to environmental stresses, cells invoke translational control to conserve resources and rapidly reprogram gene expression for optimal adaptation. A central mechanism for translational control involves phosphorylation of the α subunit of eIF2 (p-eIF2α), which reduces delivery of initiator tRNA to ribosomes. Because p-eIF2α is invoked by multiple protein kinases, each responding to distinct stresses, this pathway is named the integrated stress response (ISR). While p-eIF2α lowers bulk translation initiation, many stress-related mRNAs are preferentially translated.Analysis of a degron-containing reporter protein GFP-CL1 reveals a role for SUMO1 in cytosolic protein quality control
Misfolded proteins are recognized and degraded through protein quality control (PQC) pathways, which are essential for maintaining proteostasis and normal cellular functions. Defects in PQC can result in disease, including cancer, cardiovascular disease, and neurodegeneration. The small ubiquitin-related modifiers (SUMOs) were previously implicated in the degradation of nuclear misfolded proteins, but their functions in cytoplasmic PQC are unclear. Here, in a systematic screen of SUMO protein mutations in the budding yeast Saccharomyces cerevisiae, we identified a mutant allele (Smt3-K38A/K40A) that sensitizes cells to proteotoxic stress induced by amino acid analogs.Elucidation of ubiquitin-conjugating enzymes that interact with RBR-type ubiquitin ligases using a liquid–liquid phase separation–based method
RING-between RING (RBR)-type ubiquitin (Ub) ligases (E3s) such as Parkin receive Ub from Ub-conjugating enzymes (E2s) in response to ligase activation. However, the specific E2s that transfer Ub to each RBR-type ligase are largely unknown because of insufficient methods for monitoring their interaction. To address this problem, we have developed a method that detects intracellular interactions between E2s and activated Parkin. Fluorescent homotetramer Azami-Green fused with E2 and oligomeric Ash (Assembly helper) fused with Parkin form a liquid–liquid phase separation (LLPS) in cells only when E2 and Parkin interact.Free fatty acids stabilize integrin β1 via S-nitrosylation to promote monocyte–endothelial adhesion
Hyperlipidemia characterized by high blood levels of free fatty acids (FFAs) is important for the progression of inflammatory cardiovascular diseases. Integrin β1 is a transmembrane receptor that drives various cellular functions, including differentiation, migration, and phagocytosis. However, the underlying mechanisms modifying integrin β1 protein and activity in mediating monocyte/macrophage adhesion to endothelium remain poorly understood. In this study, we demonstrated that integrin β1 protein underwent S-nitrosylation in response to nitrosative stress in macrophages.Deep mutational analysis of elongation factor eEF2 residues implicated in human disease to identify functionally important contacts with the ribosome
An emerging body of research is revealing mutations in elongation factor eEF2 that are implicated in both inherited and de novo neurodevelopmental disorders. Previous structural analysis has revealed that most pathogenic amino acid substitutions map to the three main points of contact between eEF2 and critical large subunit rRNA elements of the ribosome, specifically to contacts with Helix 69, Helix 95, also known as the sarcin-ricin loop, and Helix 43 of the GTPase-associated center. In order to further investigate these eEF2–ribosome interactions, we identified a series of yeast eEF2 amino acid residues based on their proximity to these functionally important rRNA elements.Diptoindonesin G is a middle domain HSP90 modulator for cancer treatment
HSP90 inhibitors can target many oncoproteins simultaneously, but none have made it through clinical trials due to dose-limiting toxicity and induction of heat shock response, leading to clinical resistance. We identified diptoindonesin G (dip G) as an HSP90 modulator that can promote degradation of HSP90 clients by binding to the middle domain of HSP90 (Kd = 0.13 ± 0.02 μM) without inducing heat shock response. This is likely because dip G does not interfere with the HSP90–HSF1 interaction like N-terminal inhibitors, maintaining HSF1 in a transcriptionally silent state.Differential contributions of the proteasome, autophagy, and chaperones to the clearance of arsenite-induced protein aggregates in yeast
The poisonous metalloid arsenite induces widespread misfolding and aggregation of nascent proteins in vivo, and this mode of toxic action might underlie its suspected role in the pathology of certain protein misfolding diseases. Evolutionarily conserved protein quality-control systems protect cells against arsenite-mediated proteotoxicity, and herein, we systematically assessed the contribution of the ubiquitin-proteasome system, the autophagy-vacuole pathway, and chaperone-mediated disaggregation to the clearance of arsenite-induced protein aggregates in Saccharomyces cerevisiae.A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of proinflammatory interleukin 12 family cytokines
Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and β subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate β subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Here, we site-specifically introduce photocrosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone–client complexes for mass spectrometry.A method for the isolation and characterization of autophagic bodies from yeast provides a key tool to investigate cargos of autophagy
Autophagy is a major cellular degradation pathway that is highly conserved among eukaryotes. The identification of cargos captured by autophagosomes is critical to our understanding of the physiological significance of autophagy in cells, but these studies can be challenging because autophagosomes disintegrate easily. In the yeast Saccharomyces cerevisiae, cells deficient in the vacuolar lipase Atg15 accumulate autophagic bodies (ABs) within the vacuole following the induction of autophagy. As ABs contain cytosolic components including proteins, RNAs, and lipids, their purification allows the identification of material targeted by autophagy for degradation.Heat shock protein Hspa13 regulates endoplasmic reticulum and cytosolic proteostasis through modulation of protein translocation
Most eukaryotic secretory proteins are cotranslationally translocated through Sec61 into the endoplasmic reticulum (ER). Because these proteins have evolved to fold in the ER, their mistargeting is associated with toxicity. Genetic experiments have implicated the ER heat shock protein 70 (Hsp70) Hspa13/STCH as involved in processing of nascent secretory proteins. Herein, we evaluate the role of Hspa13 in protein import and the maintenance of cellular proteostasis in human cells, primarily using the human embryonic kidney 293T cell line.Structure of the drug target ClpC1 unfoldase in action provides insights on antibiotic mechanism of action
The unfoldase ClpC1 is one of the most exciting drug targets against tuberculosis. This AAA+ unfoldase works in cooperation with the ClpP1P2 protease and is the target of at least four natural product antibiotics: cyclomarin, ecumicin, lassomycin, and rufomycin. Although these molecules are promising starting points for drug development, their mechanisms of action remain largely unknown. Taking advantage of a middle domain mutant, we determined the first structure of Mycobacterium tuberculosis ClpC1 in its apo, cyclomarin-, and ecumicin-bound states via cryo-EM.α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α
α-synucleinopathy is driven by an imbalance of synthesis and degradation of α-synuclein (αSyn), causing a build up of αSyn aggregates and post-translationally modified species, which not only interfere with normal cellular metabolism but also by their secretion propagates the disease. Therefore, a better understanding of αSyn degradation pathways is needed to address α-synucleinopathy. Here, we used the nerve growth factor–differentiated catecholaminergic PC12 neuronal cell line, which was conferred α-synucleinopathy by inducible expression of αSyn and tubulin polymerization-promoting protein p25α.Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 in helix-3 affect ubiquitination activity and decrease enzyme stability
Rad6, an E2 ubiquitin-conjugating enzyme conserved from yeast to humans, functions in transcription, genome maintenance, and proteostasis. The contributions of many conserved secondary structures of Rad6 and its human homologs UBE2A and UBE2B to their biological functions are not understood. A mutant RAD6 allele with a missense substitution at alanine-126 (A126) of helix-3 that causes defects in telomeric gene silencing, DNA repair, and protein degradation was reported over 2 decades ago. Here, using a combination of genetics, biochemical, biophysical, and computational approaches, we discovered that helix-3 A126 mutations compromise the ability of Rad6 to ubiquitinate target proteins without disrupting interactions with partner E3 ubiquitin-ligases that are required for their various biological functions in vivo.Rate-limiting hydrolysis in ribosomal release reactions revealed by ester activation
Translation terminates by releasing the polypeptide chain in one of two chemical reactions catalyzed by the ribosome. Release is also a target for engineering, as readthrough of a stop codon enables incorporation of unnatural amino acids and treatment of genetic diseases. Hydrolysis of the ester bond of peptidyl-tRNA requires conformational changes of both a class I release factor (RF) protein and the peptidyl transferase center of a large subunit rRNA. The rate-limiting step was proposed to be hydrolysis at physiological pH and an RF conformational change at higher pH, but evidence was indirect.A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability
The IL-3, IL-5, and GM-CSF family of cytokines play an essential role in the growth, differentiation, and effector functions of multiple hematopoietic cell types. Receptors in this family are composed of cytokine-specific α chains and a common β chain (CSF2RB), responsible for the majority of downstream signaling. CSF2RB abundance and stability influence the magnitude of the cellular response to cytokine stimulation, but the exact mechanisms of regulation are not well understood. Here, we use genetic screens in multiple cellular contexts and cytokine conditions to identify STUB1, an E3 ubiquitin ligase, and CHIC2 as regulators of CSF2RB ubiquitination and protein stability.Single molecule microscopy reveals diverse actions of substrate sequences that impair ClpX AAA+ ATPase function
AAA+ (ATPases Associated with diverse cellular Activities) proteases unfold substrate proteins by pulling the substrate polypeptide through a narrow pore. To overcome the barrier to unfolding, substrates may require extended association with the ATPase. Failed unfolding attempts can lead to a slip of grip, which may result in substrate dissociation, but how substrate sequence affects slippage is unresolved. Here, we measured single molecule dwell time using total internal reflection fluorescence microscopy, scoring time-dependent dissociation of engaged substrates from bacterial AAA+ ATPase unfoldase/translocase ClpX.Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors
Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system. Maintenance of GABAA receptor protein homeostasis (proteostasis) in cells utilizing its interacting proteins is essential for the function of GABAA receptors. However, how the proteostasis network orchestrates GABAA receptor biogenesis in the endoplasmic reticulum is not well understood. Here, we employed a proteomics-based approach to systematically identify the interactomes of GABAA receptors.Human eukaryotic initiation factor 4E (eIF4E) and the nucleotide-bound state of eIF4A regulate eIF4F binding to RNA
During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA.The amino acid sensor GCN2 suppresses terminal oligopyrimidine (TOP) mRNA translation via La-related protein 1 (LARP1)
La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5′TOP motif, resulting in the displacement of the cap-binding protein eIF4E from TOP mRNAs. However, the involvement of additional signaling pathway in regulating LARP1-mediated inhibition of TOP mRNA translation is largely unexplored.Proteasome activator 28γ (PA28γ) allosterically activates trypsin-like proteolysis by binding to the α-ring of the 20S proteasome
Proteasome activator 28γ (PA28γ/REGγ) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and implicated in various diseases, including certain cancers and systemic lupus erythematosus. Despite years of investigation, how PA28γ functions to stimulate proteasomal protein degradation remains unclear. Alternative hypotheses have been proposed for the molecular mechanism of PA28γ, including the following: (1) substrate selection, (2) allosteric upregulation of the trypsin-like (T-L) site, (3) allosteric inhibition of the chymotrypsin-like (CT-L) and caspase-like (C-L) sites, (4) conversion of the CT-L or C-L sites to new T-L sites, and (5) gate opening alone or in combination with a previous hypothesis.
