An expanded lexicon for the ubiquitin code.

Our understanding of the ubiquitin code has greatly evolved from conventional E1, E2 and E3 enzymes that modify Lys residues on specific substrates with a single type of ubiquitin chain to more complex processes that regulate and mediate ubiquitylation. In this Review, we discuss recently discovered endogenous mechanisms and unprecedented pathways by which pathogens rewrite the ubiquitin code to promote infection. These processes include unconventional ubiquitin modifications involving ester linkages with proteins, lipids and sugars, or ubiquitylation through a phosphoribosyl bridge involving Arg42 of ubiquitin. We also introduce the enzymatic pathways that write and reverse these modifications, such as the papain-like proteases of severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Furthermore, structural studies have revealed that the ultimate functions of ubiquitin are mediated not simply by straightforward recognition by ubiquitin-binding domains. Instead, elaborate multivalent interactions between ubiquitylated targets or ubiquitin chains and their readers (for example, the proteasome, the MLL1 complex or DOT1L) can elicit conformational changes that regulate protein degradation or transcription. The newly discovered mechanisms provide opportunities for innovative therapeutic interventions for diseases such as cancer and infectious diseases.

Proteasomal and Autophagic Degradation Systems.

Autophagy and the ubiquitin-proteasome system are the two major quality control pathways responsible for cellular homeostasis. As such, they provide protection against age-associated changes and a plethora of human diseases. Ubiquitination is utilized as a degradation signal by both systems, albeit in different ways, to mark cargoes for proteasomal and lysosomal degradation. Both systems intersect and communicate at multiple points to coordinate their actions in proteostasis and organelle homeostasis. This review summarizes molecular details of how proteasome and autophagy pathways are functionally interconnected in cells and indicates common principles and nodes of communication that can be therapeutically exploited.

Autophagy Captures the Nobel Prize.

This year's Nobel Prize in Physiology or Medicine has been awarded to Yoshinori Ohsumi for the discovery of the molecular principles governing autophagy, an intracellular degradation pathway routed via lysosomes or vacuoles. It is a story of a simple yet insightful yeast genetic screen that revealed the inner circuitry of one of the most powerful quality-control pathways in cells.

Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination.

Conventional ubiquitination involves the ATP-dependent formation of amide bonds between the ubiquitin C terminus and primary amines in substrate proteins. Recently, SdeA, an effector protein of pathogenic Legionella pneumophila, was shown to mediate NAD-dependent and ATP-independent ubiquitin transfer to host proteins. Here, we identify a phosphodiesterase domain in SdeA that efficiently catalyzes phosphoribosylation of ubiquitin on a specific arginine via an ADP-ribose-ubiquitin intermediate. SdeA also catalyzes a chemically and structurally distinct type of substrate ubiquitination by conjugating phosphoribosylated ubiquitin to serine residues of protein substrates via a phosphodiester bond. Furthermore, phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes of the conventional ubiquitination cascade, thereby impairing numerous cellular processes including mitophagy, TNF signaling, and proteasomal degradation. We propose that phosphoribosylation of ubiquitin potently modulates ubiquitin functions in mammalian cells.

Mechanism and medical implications of mammalian autophagy.

Autophagy is a highly conserved catabolic process induced under various conditions of cellular stress, which prevents cell damage and promotes survival in the event of energy or nutrient shortage and responds to various cytotoxic insults. Thus, autophagy has primarily cytoprotective functions and needs to be tightly regulated to respond correctly to the different stimuli that cells experience, thereby conferring adaptation to the ever-changing environment. It is now apparent that autophagy is deregulated in the context of various human pathologies, including cancer and neurodegeneration, and its modulation has considerable potential as a therapeutic approach.

Targeted protein degradation via intramolecular bivalent glues.

Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

ER remodeling via ER-phagy.

The endoplasmic reticulum (ER) is a hotspot for many essential cellular functions. The ER membrane is highly dynamic, which affects many cellular processes that take place within the ER. One such process is ER-phagy, a selective degradation of ER fragments (including membranes and luminal content), which serves to preserve the size of ER while adapting its morphology under basal and stress conditions. In order to be degraded, the ER undergoes selective fragmentation facilitated by specialized ER-shaping proteins that also act as ER-phagy receptors. Their ability to sense and induce membrane curvature, as well as to bridge the ER with autophagy machinery, allows for a successful ER fragmentation and delivery of these fragments to the lysosome for degradation and recycling. In this review, we provide insights into ER-phagy from the perspective of membrane remodeling. We highlight the importance of ER membrane dynamics during ER-phagy and emphasize how its dysregulation reflects on human physiology and pathology.

Mycobacterium tuberculosis hijacks ubiquitin to inhibit pyroptosis.

Chai et al.1 reveal that the eukaryotic-like effector protein PtpB from Mycobacterium tuberculosis (MTB) dephosphorylates phospholipid membrane proteins, which prevents membrane localization of cleaved gasdermin D, inhibiting pyroptosis and cytokine release by infected macrophages to enable MTB immune evasion.

Deciphering functions of branched ubiquitin chains.

The anaphase-promoting complex/cyclosome targets proteins for degradation by catalyzing homotypic ubiquitin chains of different linkage types. In this issue of Cell, Meyer and Rape diversify the degradation signals by demonstrating that the APC/C and its cognate E2 conjugating enzymes enhance the rate of substrate degradation by decorating them with branched Lys11 and Lys48 ubiquitin chains.

Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.

The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.

Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.

Cullins getting undressed by the protein exchange factor Cand1.

Cullin-RING ubiquitin ligase complexes (CRLs) rely on a vast array of adaptor proteins to recognize their substrates. Pierce et al. and related papers from Zemla et al. and Wu et al. in Nature Communications show that Cand1 promotes exchange of adaptor proteins to regulate the CRL repertoire.

Regulation of Phosphoribosyl-Linked Serine Ubiquitination by Deubiquitinases DupA and DupB.

The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection.

Ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions.

Ubiquitin acts as a versatile cellular signal that controls a wide range of biological processes including protein degradation, DNA repair, endocytosis, autophagy, transcription, immunity, and inflammation. The specificity of ubiquitin signaling is achieved by alternative conjugation signals (monoubiquitin and ubiquitin chains) and interactions with ubiquitin-binding proteins (known as ubiquitin receptors) that decode ubiquitinated target signals into biochemical cascades in the cell. Herein, we review the current knowledge pertaining to the structural and functional features of ubiquitin-binding proteins and the mechanisms by which they recognize various types of ubiquitin topologies. The combinatorial use of diverse ubiquitin-binding domains (UBDs) in full-length proteins, selective recognition of chains with distinct linkages and length, and posttranslational modifications of ubiquitin receptors or multivalent interactions within protein complexes illustrate a few mechanisms by which a circuitry of signaling networks can be rewired by ubiquitin-binding proteins to control cellular functions in vivo.

Regulation of Host-Pathogen Interactions via the Ubiquitin System.

Ubiquitination is a posttranslational modification that regulates a multitude of cellular functions. Pathogens, such as bacteria and viruses, have evolved sophisticated mechanisms that evade or counteract ubiquitin-dependent host responses, or even exploit the ubiquitin system to their own advantage. This is largely done by numerous pathogen virulence factors that encode E3 ligases and deubiquitinases, which are often used as weapons in pathogen-host cell interactions. Moreover, upon pathogen attack, host cellular signaling networks undergo major ubiquitin-dependent changes to protect the host cell, including coordination of innate immunity, remodeling of cellular organelles, reorganization of the cytoskeleton, and reprogramming of metabolic pathways to restrict growth of the pathogen. Here we provide mechanistic insights into ubiquitin regulation of host-pathogen interactions and how it affects bacterial and viral pathogenesis and the organization and response of the host cell.

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

The mission to ensure continued funding for excellent basic research.

The surprising decision by Novo Nordisk Foundation (NNF) to discontinue funding for the Center for Protein Research in Copenhagen should prompt discussions about public and private commitment to support basic research.

Not all autophagy membranes are created equal.

During starvation, the cell recycles cytoplasmic material by sequestering it in double-membrane organelles, called autophagosomes, which eventually fuse with lysosomes. In this issue, Hailey et al. (2010) identify the outer membrane of mitochondria as a new source of autophagosomal membranes during starvation.