ATP-Dependent Steps in Peroxisomal Protein Import.

Peroxisomes are organelles that play a central role in lipid metabolism and cellular redox homeostasis. The import of peroxisomal matrix proteins by peroxisomal targeting signal (PTS) receptors is an ATP-dependent mechanism. However, the energy-dependent steps do not occur early during the binding of the receptor-cargo complex to the membrane but late, because they are linked to the peroxisomal export complex for the release of the unloaded receptor. The first ATP-demanding step is the cysteine-dependent monoubiquitination of the PTS receptors, which is required for recognition by the AAA+ peroxins. They execute the second ATP-dependent step by extracting the ubiqitinated PTS receptors from the membrane for release back to the cytosol. After deubiquitination, the PTS receptors regain import competence and can facilitate further rounds of cargo import. Here, we give a general overview and discuss recent data regarding the ATP-dependent steps in peroxisome protein import.

MTFP1 controls mitochondrial fusion to regulate inner membrane quality control and maintain mtDNA levels.

Mitochondrial dynamics play a critical role in cell fate decisions and in controlling mtDNA levels and distribution. However, the molecular mechanisms linking mitochondrial membrane remodeling and quality control to mtDNA copy number (CN) regulation remain elusive. Here, we demonstrate that the inner mitochondrial membrane (IMM) protein mitochondrial fission process 1 (MTFP1) negatively regulates IMM fusion. Moreover, manipulation of mitochondrial fusion through the regulation of MTFP1 levels results in mtDNA CN modulation. Mechanistically, we found that MTFP1 inhibits mitochondrial fusion to isolate and exclude damaged IMM subdomains from the rest of the network. Subsequently, peripheral fission ensures their segregation into small MTFP1-enriched mitochondria (SMEM) that are targeted for degradation in an autophagic-dependent manner. Remarkably, MTFP1-dependent IMM quality control is essential for basal nucleoid recycling and therefore to maintain adequate mtDNA levels within the cell.

Mechanism of orphan subunit recognition during assembly quality control.

Cells contain numerous abundant molecular machines assembled from multiple subunits. Imbalances in subunit production and failed assembly generate orphan subunits that are eliminated by poorly defined pathways. Here, we determined how orphan subunits of the cytosolic chaperonin CCT are recognized. Several unassembled CCT subunits recruited the E3 ubiquitin ligase HERC2 using ZNRD2 as an adaptor. Both factors were necessary for orphan CCT subunit degradation in cells, sufficient for CCT subunit ubiquitination with purified factors, and necessary for optimal cell fitness. Domain mapping and structure prediction defined the molecular features of a minimal HERC2-ZNRD2-CCT module. The structural model, whose key elements were validated in cells using point mutants, shows why ZNRD2 selectively recognizes multiple orphaned CCT subunits without engaging assembled CCT. Our findings reveal how failures during CCT assembly are monitored and provide a paradigm for the molecular recognition of orphan subunits, the largest source of quality control substrates in cells.

Orphan quality control shapes network dynamics and gene expression.

All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.

Quality control and analytic best practices for testing genetic models of sex differences in large populations.

Phenotypic sex-based differences exist for many complex traits. In other cases, phenotypes may be similar, but underlying biology may vary. Thus, sex-aware genetic analyses are becoming increasingly important for understanding the mechanisms driving these differences. To this end, we provide a guide outlining the current best practices for testing various models of sex-dependent genetic effects in complex traits and disease conditions, noting that this is an evolving field. Insights from sex-aware analyses will not only teach us about the biology of complex traits but also aid in achieving the goals of precision medicine and health equity for all.

Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival.

The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.

An E3 ligase network engages GCN1 to promote the degradation of translation factors on stalled ribosomes.

Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.

Quality Control of Procollagen in Cells.

Collagen is the most abundant protein in mammals. A unique feature of collagen is its triple-helical structure formed by the Gly-Xaa-Yaa repeats. Three single chains of procollagen make a trimer, and the triple-helical structure is then folded in the endoplasmic reticulum (ER). This unique structure is essential for collagen's functions in vivo, including imparting bone strength, allowing signal transduction, and forming basement membranes. The triple-helical structure of procollagen is stabilized by posttranslational modifications and intermolecular interactions, but collagen is labile even at normal body temperature. Heat shock protein 47 (Hsp47) is a collagen-specific molecular chaperone residing in the ER that plays a pivotal role in collagen biosynthesis and quality control of procollagen in the ER. Mutations that affect the triple-helical structure or result in loss of Hsp47 activity cause the destabilization of procollagen, which is then degraded by autophagy. In this review, we present the current state of the field regarding quality control of procollagen.

Mitocytosis, a migrasome-mediated mitochondrial quality-control process.

Damaged mitochondria need to be cleared to maintain the quality of the mitochondrial pool. Here, we report mitocytosis, a migrasome-mediated mitochondrial quality-control process. We found that, upon exposure to mild mitochondrial stresses, damaged mitochondria are transported into migrasomes and subsequently disposed of from migrating cells. Mechanistically, mitocytosis requires positioning of damaged mitochondria at the cell periphery, which occurs because damaged mitochondria avoid binding to inward motor proteins. Functionally, mitocytosis plays an important role in maintaining mitochondrial quality. Enhanced mitocytosis protects cells from mitochondrial stressor-induced loss of mitochondrial membrane potential (MMP) and mitochondrial respiration; conversely, blocking mitocytosis causes loss of MMP and mitochondrial respiration under normal conditions. Physiologically, we demonstrate that mitocytosis is required for maintaining MMP and viability in neutrophils in vivo. We propose that mitocytosis is an important mitochondrial quality-control process in migrating cells, which couples mitochondrial homeostasis with cell migration.

Physiological Functions of Intracellular Protein Degradation.

While cellular proteins were initially thought to be stable, research over the last decades has firmly established that intracellular protein degradation is an active and highly regulated process: Lysosomal, proteasomal, and mitochondrial degradation systems were identified and found to be involved in a staggering number of biological functions. Here, we provide a global overview of the diverse roles of cellular protein degradation using seven categories: homeostasis, regulation, quality control, stoichiometry control, proteome remodeling, immune surveillance, and baseline turnover. Using selected examples, we outline how proteins are degraded and why this is functionally relevant.

Mitochondrial Proteases: Multifaceted Regulators of Mitochondrial Plasticity.

Mitochondria are essential metabolic hubs that dynamically adapt to physiological demands. More than 40 proteases residing in different compartments of mitochondria, termed mitoproteases, preserve mitochondrial proteostasis and are emerging as central regulators of mitochondrial plasticity. These multifaceted enzymes limit the accumulation of short-lived, regulatory proteins within mitochondria, modulate the activity of mitochondrial proteins by protein processing, and mediate the degradation of damaged proteins. Various signaling cascades coordinate the activity of mitoproteases to preserve mitochondrial homeostasis and ensure cell survival. Loss of mitoproteases severely impairs the functional integrity of mitochondria, is associated with aging, and causes pleiotropic diseases. Understanding the dual function of mitoproteases as regulatory and quality control enzymes will help unravel the role of mitochondrial plasticity in aging and disease.

Standing on the Shoulders of Viruses.

My coworkers and I have used animal viruses and their interaction with host cells to investigate cellular processes difficult to study by other means. This approach has allowed us to branch out in many directions, including membrane protein characterization, endocytosis, secretion, protein folding, quality control, and glycobiology. At the same time, our aim has been to employ cell biological approaches to expand the fundamental understanding of animal viruses and their pathogenic lifestyles. We have studied mechanisms of host cell entry and the uncoating of incoming viruses as well as the synthesis, folding, maturation, and intracellular movement of viral proteins and molecular assemblies. I have had the privilege to work in institutions in four different countries. The early years in Finland (the University of Helsinki) were followed by 6 years in Germany (European Molecular Biology Laboratory), 16 years in the United States (Yale School of Medicine), and 16 years in Switzerland (ETH Zurich).

Chemical Biology Framework to Illuminate Proteostasis.

Protein folding in the cell is mediated by an extensive network of >1,000 chaperones, quality control factors, and trafficking mechanisms collectively termed the proteostasis network. While the components and organization of this network are generally well established, our understanding of how protein-folding problems are identified, how the network components integrate to successfully address challenges, and what types of biophysical issues each proteostasis network component is capable of addressing remains immature. We describe a chemical biology-informed framework for studying cellular proteostasis that relies on selection of interesting protein-folding problems and precise researcher control of proteostasis network composition and activities. By combining these methods with multifaceted strategies to monitor protein folding, degradation, trafficking, and aggregation in cells, researchers continue to rapidly generate new insights into cellular proteostasis.

Alanine Tails Signal Proteolysis in Bacterial Ribosome-Associated Quality Control.

In ribosome-associated quality control (RQC), Rqc2/NEMF closely supports the E3 ligase Ltn1/listerin in promoting ubiquitylation and degradation of aberrant nascent-chains obstructing large (60S) ribosomal subunits-products of ribosome stalling during translation. However, while Ltn1 is eukaryote-specific, Rqc2 homologs are also found in bacteria and archaea; whether prokaryotic Rqc2 has an RQC-related function has remained unknown. Here, we show that, as in eukaryotes, a bacterial Rqc2 homolog (RqcH) recognizes obstructed 50S subunits and promotes nascent-chain proteolysis. Unexpectedly, RqcH marks nascent-chains for degradation in a direct manner, by appending C-terminal poly-alanine tails that act as degrons recognized by the ClpXP protease. Furthermore, RqcH acts redundantly with tmRNA/ssrA and protects cells against translational and environmental stresses. Our results uncover a proteolytic-tagging mechanism with implications toward the function of related modifications in eukaryotes and suggest that RQC was already active in the last universal common ancestor (LUCA) to help cope with incomplete translation.

Protein Sequence Editing of SKN-1A/Nrf1 by Peptide:N-Glycanase Controls Proteasome Gene Expression.

The proteasome mediates selective protein degradation and is dynamically regulated in response to proteotoxic challenges. SKN-1A/Nrf1, an endoplasmic reticulum (ER)-associated transcription factor that undergoes N-linked glycosylation, serves as a sensor of proteasome dysfunction and triggers compensatory upregulation of proteasome subunit genes. Here, we show that the PNG-1/NGLY1 peptide:N-glycanase edits the sequence of SKN-1A protein by converting particular N-glycosylated asparagine residues to aspartic acid. Genetically introducing aspartates at these N-glycosylation sites bypasses the requirement for PNG-1/NGLY1, showing that protein sequence editing rather than deglycosylation is key to SKN-1A function. This pathway is required to maintain sufficient proteasome expression and activity, and SKN-1A hyperactivation confers resistance to the proteotoxicity of human amyloid beta peptide. Deglycosylation-dependent protein sequence editing explains how ER-associated and cytosolic isoforms of SKN-1 perform distinct cytoprotective functions corresponding to those of mammalian Nrf1 and Nrf2. Thus, we uncover an unexpected mechanism by which N-linked glycosylation regulates protein function and proteostasis.

Protein Quality Control Degradation in the Nucleus.

Nuclear proteins participate in diverse cellular processes, many of which are essential for cell survival and viability. To maintain optimal nuclear physiology, the cell employs the ubiquitin-proteasome system to eliminate damaged and misfolded proteins in the nucleus that could otherwise harm the cell. In this review, we highlight the current knowledge about the major ubiquitin-protein ligases involved in protein quality control degradation (PQCD) in the nucleus and how they orchestrate their functions to eliminate misfolded proteins in different nuclear subcompartments. Many human disorders are causally linked to protein misfolding in the nucleus, hence we discuss major concepts that still need to be clarified to better understand the basis of the nuclear misfolded proteins' toxic effects. Additionally, we touch upon potential strategies for manipulating nuclear PQCD pathways to ameliorate diseases associated with protein misfolding and aggregation in the nucleus.

Protein Quality Control of the Endoplasmic Reticulum and Ubiquitin-Proteasome-Triggered Degradation of Aberrant Proteins: Yeast Pioneers the Path.

Cells must constantly monitor the integrity of their macromolecular constituents. Proteins are the most versatile class of macromolecules but are sensitive to structural alterations. Misfolded or otherwise aberrant protein structures lead to dysfunction and finally aggregation. Their presence is linked to aging and a plethora of severe human diseases. Thus, misfolded proteins have to be rapidly eliminated. Secretory proteins constitute more than one-third of the eukaryotic proteome. They are imported into the endoplasmic reticulum (ER), where they are folded and modified. A highly elaborated machinery controls their folding, recognizes aberrant folding states, and retrotranslocates permanently misfolded proteins from the ER back to the cytosol. In the cytosol, they are degraded by the highly selective ubiquitin-proteasome system. This process of protein quality control followed by proteasomal elimination of the misfolded protein is termed ER-associated degradation (ERAD), and it depends on an intricate interplay between the ER and the cytosol.

Target-Based Discovery of an Inhibitor of the Regulatory Phosphatase PPP1R15B.

Protein phosphorylation is a prevalent and ubiquitous mechanism of regulation. Kinases are popular drug targets, but identifying selective phosphatase inhibitors has been challenging. Here, we used surface plasmon resonance to design a method to enable target-based discovery of selective serine/threonine phosphatase inhibitors. The method targeted a regulatory subunit of protein phosphatase 1, PPP1R15B (R15B), a negative regulator of proteostasis. This yielded Raphin1, a selective inhibitor of R15B. In cells, Raphin1 caused a rapid and transient accumulation of its phosphorylated substrate, resulting in a transient attenuation of protein synthesis. In vitro, Raphin1 inhibits the recombinant R15B-PP1c holoenzyme, but not the closely related R15A-PP1c, by interfering with substrate recruitment. Raphin1 was orally bioavailable, crossed the blood-brain barrier, and demonstrated efficacy in a mouse model of Huntington's disease. This identifies R15B as a druggable target and provides a platform for target-based discovery of inhibitors of serine/threonine phosphatases.

Protein Misfolding, Amyloid Formation, and Human Disease: A Summary of Progress Over the Last Decade.

Peptides and proteins have been found to possess an inherent tendency to convert from their native functional states into intractable amyloid aggregates. This phenomenon is associated with a range of increasingly common human disorders, including Alzheimer and Parkinson diseases, type II diabetes, and a number of systemic amyloidoses. In this review, we describe this field of science with particular reference to the advances that have been made over the last decade in our understanding of its fundamental nature and consequences. We list the proteins that are known to be deposited as amyloid or other types of aggregates in human tissues and the disorders with which they are associated, as well as the proteins that exploit the amyloid motif to play specific functional roles in humans. In addition, we summarize the genetic factors that have provided insight into the mechanisms of disease onset. We describe recent advances in our knowledge of the structures of amyloid fibrils and their oligomeric precursors and of the mechanisms by which they are formed and proliferate to generate cellular dysfunction. We show evidence that a complex proteostasis network actively combats protein aggregation and that such an efficient system can fail in some circumstances and give rise to disease. Finally, we anticipate the development of novel therapeutic strategies with which to prevent or treat these highly debilitating and currently incurable conditions.

The Upsides and Downsides of Organelle Interconnectivity.

Interconnectivity and feedback control are hallmarks of biological systems. This includes communication between organelles, which allows them to function and adapt to changing cellular environments. While the specific mechanisms for all communications remain opaque, unraveling the wiring of organelle networks is critical to understand how biological systems are built and why they might collapse, as occurs in aging. A comprehensive understanding of all the routes involved in inter-organelle communication is still lacking, but important themes are beginning to emerge, primarily in budding yeast. These routes are reviewed here in the context of sub-system proteostasis and complex adaptive systems theory.