Global, site-resolved analysis of ubiquitylation occupancy and turnover rate reveals systems properties.

Ubiquitylation regulates most proteins and biological processes in a eukaryotic cell. However, the site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have not been quantified. Here we present an integrated picture of the global ubiquitylation site occupancy and half-life. Ubiquitylation site occupancy spans over four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and regulation of sites by proteasome inhibitors are strongly interrelated, and these attributes distinguish sites involved in proteasomal degradation and cellular signaling. Sites in structured protein regions exhibit longer half-lives and stronger upregulation by proteasome inhibitors than sites in unstructured regions. Importantly, we discovered a surveillance mechanism that rapidly and site-indiscriminately deubiquitylates all ubiquitin-specific E1 and E2 enzymes, protecting them against accumulation of bystander ubiquitylation. The work provides a systems-scale, quantitative view of ubiquitylation properties and reveals general principles of ubiquitylation-dependent governance.

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.

Gene Expression Changes and Community Turnover Differentially Shape the Global Ocean Metatranscriptome.

Ocean microbial communities strongly influence the biogeochemistry, food webs, and climate of our planet. Despite recent advances in understanding their taxonomic and genomic compositions, little is known about how their transcriptomes vary globally. Here, we present a dataset of 187 metatranscriptomes and 370 metagenomes from 126 globally distributed sampling stations and establish a resource of 47 million genes to study community-level transcriptomes across depth layers from pole-to-pole. We examine gene expression changes and community turnover as the underlying mechanisms shaping community transcriptomes along these axes of environmental variation and show how their individual contributions differ for multiple biogeochemically relevant processes. Furthermore, we find the relative contribution of gene expression changes to be significantly lower in polar than in non-polar waters and hypothesize that in polar regions, alterations in community activity in response to ocean warming will be driven more strongly by changes in organismal composition than by gene regulatory mechanisms. VIDEO ABSTRACT.

Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis.

Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed "multiplexed proteome dynamics profiling" (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems.

Intrinsically determined turnover underlies broad heterogeneity in plasma-cell lifespan.

Antibodies produced by antibody-secreting plasma cells (ASCs) underlie multiple forms of long-lasting immunity. Here we examined the mechanisms regulating ASC turnover and persistence using a genetic reporter to time-stamp ASCs. This approach revealed ASC lifespans as heterogeneous and falling on a continuum, with only a small fraction surviving for >60 days. ASC longevity past 60 days was independent of isotype but correlated with a phenotype that developed progressively and ultimately associated with an underlying "long-lived" ASC (LL ASC)-enriched transcriptional program. While some of the differences between LL ASCs and other ASCs appeared to be acquired with age, other features were shared with some younger ASCs, such as high CD138 and CD93. Turnover was unaffected by altered ASC production, arguing against competition for niches as a major driver of turnover. Thus, ASC turnover is set by intrinsic lifespan limits, with steady-state population dynamics governed by niche vacancy rather than displacement.

The Logic of the 26S Proteasome.

The ubiquitin proteasome pathway is responsible for most of the protein degradation in mammalian cells. Rates of degradation by this pathway have generally been assumed to be determined by rates of ubiquitylation. However, recent studies indicate that proteasome function is also tightly regulated and determines whether a ubiquitylated protein is destroyed or deubiquitylated and survives longer. This article reviews recent advances in our understanding of the proteasome's multistep ATP-dependent mechanism, its biochemical and structural features that ensure efficient proteolysis and ubiquitin recycling while preventing nonselective proteolysis, and the regulation of proteasome activity by interacting proteins and subunit modifications, especially phosphorylation.

Protein degradation on the global scale.

Protein degradation occurs through proteasomal, endosomal, and lysosomal pathways. Technological advancements have allowed for the determination of protein copy numbers and turnover rates on a global scale, which has provided an overview of trends and rules governing protein degradation. Sharper chemical and gene-editing tools have enabled the specific perturbation of each degradation pathway, whose effects on protein dynamics can now be comprehensively analyzed. We review major studies and innovation in this field and discuss the interdependence between the major pathways of protein degradation.

Dissecting the Structural Dynamics of the Nuclear Pore Complex.

Cellular processes are largely carried out by macromolecular assemblies, most of which are dynamic, having components that are in constant flux. One such assembly is the nuclear pore complex (NPC), an ∼50 MDa assembly comprised of ∼30 different proteins called Nups that mediates selective macromolecular transport between the nucleus and cytoplasm. We developed a proteomics method to provide a comprehensive picture of the yeast NPC component dynamics. We discovered that, although all Nups display uniformly slow turnover, their exchange rates vary considerably. Surprisingly, this exchange rate was relatively unrelated to each Nup's position, accessibility, or role in transport but correlated with its structural role; scaffold-forming Nups exchange slowly, whereas flexible connector Nups threading throughout the NPC architecture exchange more rapidly. Targeted perturbations in the NPC structure revealed a dynamic resilience to damage. Our approach opens a new window into macromolecular assembly dynamics.

Terminal modification, sequence, length, and PIWI-protein identity determine piRNA stability.

In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal trimming and 2'-O-methylation. Both trimming and methylation influence piRNA stability. Our biochemical data show that multiple mechanisms destabilize unmethylated mouse piRNAs, depending on whether the piRNA 5' or 3' sequence is complementary to a trigger RNA. Unlike target-directed degradation of microRNAs, complementarity-dependent destabilization of piRNAs in mice and flies is blocked by 3' terminal 2'-O-methylation and does not require base pairing to both the piRNA seed and the 3' sequence. In flies, 2'-O-methylation also protects small interfering RNAs (siRNAs) from complementarity-dependent destruction. By contrast, pre-piRNA trimming protects mouse piRNAs from a degradation pathway unaffected by trigger complementarity. In testis lysate and in vivo, internal or 3' terminal uridine- or guanine-rich tracts accelerate pre-piRNA decay. Loss of both trimming and 2'-O-methylation causes the mouse piRNA pathway to collapse, demonstrating that these modifications collaborate to stabilize piRNAs.

Decapping NAD-RNAs: TIR domain-containing proteins stand out for specificity.

A recently characterized RNA modification is NAD+-modified RNAs (NAD-RNAs). Various enzymes decap NAD-RNAs, and Wang and Yu et al. now describe another, namely Toll/interleukin-1 receptor (TIR) domain-containing proteins of bacteria and Archaea. TIR decapping products are a specific variant of cyclic ADP ribose (ADPR)-RNAs (v-cADPR-RNAs), opening a new window to the NAD-RNA world.

YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus.

The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the forming autophagosome. Using quantitative proteomic analysis and two novel Golgiphagy reporter systems, we found that the five-pass transmembrane Golgi-resident proteins YIPF3 and YIPF4 constitute a Golgiphagy receptor. The interaction of this complex with LC3B, GABARAP, and GABARAPL1 is dependent on a LIR motif within YIPF3 and putative phosphorylation sites immediately upstream; the stability of the complex is governed by YIPF4. Expression of a YIPF3 protein containing a mutated LIR motif caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays reported here may be readily adapted to different experimental contexts to help deepen our understanding of Golgiphagy.

A Primase-Induced Conformational Switch Controls the Stability of the Bacterial Replisome.

Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.

Determining and interpreting protein lifetimes in mammalian tissues.

The orchestration of protein production and degradation, and the regulation of protein lifetimes, play a central role in the majority of biological processes. Recent advances in proteomics have enabled the estimation of protein half-lives for thousands of proteins in vivo. What is the utility of these measurements, and how can they be leveraged to interpret the proteome changes occurring during development, aging, and disease? This opinion article summarizes leading technical approaches and highlights their strengths and weaknesses. We also disambiguate frequently used terminology, illustrate recent mechanistic insights, and provide guidance for interpreting and validating protein turnover measurements. Overall, protein lifetimes, coupled to estimates of protein levels, are essential for obtaining a deep understanding of mammalian biology and the basic processes defining life itself.

Recycling of the actin monomer pool limits the lifetime of network turnover.

Intracellular organization is largely mediated by actin turnover. Cellular actin networks continuously assemble and disassemble, while maintaining their overall appearance. This behavior, called "dynamic steady state," allows cells to sense and adapt to their environment. However, how structural stability can be maintained during the constant turnover of a limited actin monomer pool is poorly understood. To answer this question, we developed an experimental system where polystyrene beads are propelled by an actin comet in a microwell containing a limited amount of components. We used the speed and the size of the actin comet tails to evaluate the system's monomer consumption and its lifetime. We established the relative contribution of actin assembly, disassembly, and recycling for a bead movement over tens of hours. Recycling mediated by cyclase-associated protein (CAP) is the key step in allowing the reuse of monomers for multiple assembly cycles. ATP supply and protein aging are also factors that limit the lifetime of actin turnover. This work reveals the balancing mechanism for long-term network assembly with a limited amount of building blocks.

Histone retention preserves epigenetic marks during heat stress-induced transcriptional memory in plants.

Plants often experience recurrent stressful events, for example, during heat waves. They can be primed by heat stress (HS) to improve the survival of more severe heat stress conditions. At certain genes, sustained expression is induced for several days beyond the initial heat stress. This transcriptional memory is associated with hyper-methylation of histone H3 lysine 4 (H3K4me3), but it is unclear how this is maintained for extended periods. Here, we determined histone turnover by measuring the chromatin association of HS-induced histone H3.3. Genome-wide histone turnover was not homogenous; in particular, H3.3 was retained longer at heat stress memory genes compared to HS-induced non-memory genes during the memory phase. While low nucleosome turnover retained H3K4 methylation, methylation loss did not affect turnover, suggesting that low nucleosome turnover sustains H3K4 methylation, but not vice versa. Together, our results unveil the modulation of histone turnover as a mechanism to retain environmentally mediated epigenetic modifications.

Regulation of branched versus linear Arp2/3-generated actin filaments.

Activation of the Arp2/3 complex by VCA-motif-bearing actin nucleation-promoting factors results in the formation of "daughter" actin filaments branching off the sides of pre-existing "mother" filaments. Alternatively, when stimulated by SPIN90, Arp2/3 directly nucleates "linear" actin filaments. Uncovering the similarities and differences between these two mechanisms is fundamental to understanding how actin cytoskeleton dynamics are regulated. Here, analysis of individual filaments reveals that, unexpectedly, the VCA motifs of WASP, N-WASP, and WASH destabilize existing branches, as well as SPIN90-Arp2/3 at linear filament ends. Furthermore, branch stabilizer cortactin and destabilizer GMF each have a similar impact on SPIN90-activated Arp2/3. However, unlike branch junctions, SPIN90-Arp2/3 at the ends of linear filaments is not destabilized by piconewton forces and does not become less stable with time. It thus appears that linear and branched Arp2/3-generated filaments respond similarly to the regulatory proteins we have tested, albeit with some differences, but significantly differ in their responses to aging and mechanical stress. These kinetic differences likely reflect the small conformational differences recently reported between Arp2/3 in branch junctions and linear filaments and suggest that their turnover in cells may be differently regulated.

Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria.

Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.

Nucleosome Turnover Regulates Histone Methylation Patterns over the Genome.

Recent studies have indicated that nucleosome turnover is rapid, occurring several times per cell cycle. To access the effect of nucleosome turnover on the epigenetic landscape, we investigated H3K79 methylation, which is produced by a single methyltransferase (Dot1l) with no known demethylase. Using chemical-induced proximity (CIP), we find that the valency of H3K79 methylation (mono-, di-, and tri-) is determined by nucleosome turnover rates. Furthermore, propagation of this mark is predicted by nucleosome turnover simulations over the genome and accounts for the asymmetric distribution of H3K79me toward the transcriptional unit. More broadly, a meta-analysis of other conserved histone modifications demonstrates that nucleosome turnover models predict both valency and chromosomal propagation of methylation marks. Based on data from worms, flies, and mice, we propose that the turnover of modified nucleosomes is a general means of propagation of epigenetic marks and a determinant of methylation valence.

Spartin-mediated lipid transfer facilitates lipid droplet turnover.

Lipid droplets (LDs) are organelles critical for energy storage and membrane lipid homeostasis, whose number and size are carefully regulated in response to cellular conditions. The molecular mechanisms underlying lipid droplet biogenesis and degradation, however, are not well understood. The Troyer syndrome protein spartin (SPG20) supports LD delivery to autophagosomes for turnover via lipophagy. Here, we characterize spartin as a lipid transfer protein whose transfer ability is required for LD degradation. Spartin copurifies with phospholipids and neutral lipids from cells and transfers phospholipids in vitro via its senescence domain. A senescence domain truncation that impairs lipid transfer in vitro also impairs LD turnover in cells while not affecting spartin association with either LDs or autophagosomes, supporting that spartin's lipid transfer ability is physiologically relevant. Our data indicate a role for spartin-mediated lipid transfer in LD turnover.

Activation of invariant natural killer T cells stimulates adipose tissue remodeling via adipocyte death and birth in obesity.

In obesity, adipose tissue undergoes dynamic remodeling processes such as adipocyte hypertrophy, hypoxia, immune responses, and adipocyte death. However, whether and how invariant natural killer T (iNKT) cells contribute to adipose tissue remodeling are elusive. In this study, we demonstrate that iNKT cells remove unhealthy adipocytes and stimulate the differentiation of healthy adipocytes. In obese adipose tissue, iNKT cells were abundantly found nearby dead adipocytes. FasL-positive adipose iNKT cells exerted cytotoxic effects to eliminate hypertrophic and pro-inflammatory Fas-positive adipocytes. Furthermore, in vivo adipocyte-lineage tracing mice model showed that activation of iNKT cells by alpha-galactosylceramide promoted adipocyte turnover, eventually leading to potentiation of the insulin-dependent glucose uptake ability in adipose tissue. Collectively, our data propose a novel role of adipose iNKT cells in the regulation of adipocyte turnover in obesity.