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The ability of proteins and nucleic acids to undergo liquid–liquid phase separation has recently emerged as an important molecular principle of how cells rapidly and reversibly compartmentalize their components into membrane-less organelles such as the nucleolus, processing bodies or stress granules 1 , 2 . How the assembly and turnover of these organelles are controlled, and how these biological condensates selectively recruit or release components are poorly understood. Here we show that members of the large and highly abundant family of RNA-dependent DEAD-box ATPases (DDXs) 3 are regulators of RNA-containing phase-separated organelles in prokaryotes and eukaryotes. Using in vitro reconstitution and in vivo experiments, we demonstrate that DDXs promote phase separation in their ATP-bound form, whereas ATP hydrolysis induces compartment turnover and release of RNA. This mechanism of membrane-less organelle regulation reveals a principle of cellular organization that is conserved from bacteria to humans. Furthermore, we show that DDXs control RNA flux into and out of phase-separated organelles, and thus propose that a cellular network of dynamic, DDX-controlled compartments establishes biochemical reaction centres that provide cells with spatial and temporal control of various RNA-processing steps, which could regulate the composition and fate of ribonucleoprotein particles. RNA-dependent DEAD-box ATPases (DDXs) regulate the dynamics of phase-separated organelles, with ATP-bound DDXs promoting phase separation, and ATP hydrolysis inducing compartment disassembly and RNA release.

Protease biology is intimately linked to the functional consequences of substrate cleavage events. Human caspases are a family of 12 fate-determining cysteine proteases that are best known for driving cell death, either apoptosis or pyroptosis. More recently, caspases have been shown to be involved in other cellular remodeling events as well including stem cell fate determination, spermatogenesis, and erythroid differentiation. Recent global proteomics methods enable characterization of the substrates that caspases cleave in live cells and cell extracts. The number of substrate targets identified for individual caspases can vary widely ranging from only a (few) dozen targets for caspases-4, -5, -9, and -14 to hundreds of targets for caspases-1, -2, -3, -6, -7, and -8. Proteomic studies characterizing the rates of target cleavage show that each caspase has a preferred substrate cohort that sometimes overlaps between caspases, but whose rates of cleavage vary over 500-fold within each group. Determining the functional consequences of discrete proteolytic events within the global substrate pool is a major challenge for the field. From the handful of individual targets that have been studied in detail, there are only a few so far that whose single cleavage event is capable of sparking apoptosis alone, such as cleavage of caspase-3/-7 and BIM EL , or for pyroptosis, gasdermin D. For the most part, it appears that cleavage events function cooperatively in the cell death process to generate a proteolytic synthetic lethal outcome. In contrast to apoptosis, far less is known about caspase biology in non-apoptotic cellular processes, such as cellular remodeling, including which caspases are activated, the mechanisms of their activation and deactivation, and the key substrate targets. Here we survey the progress made in global identification of caspase substrates using proteomics and the exciting new avenues these studies have opened for understanding the molecular logic of substrate cleavage in apoptotic and non-apoptotic processes.

Cyclin-dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different cyclin–CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs) 1 , 2 . However, the principles on which cyclin–CDK complexes organize the temporal order of cell cycle events are contentious 3 . One model proposes that S-CDKs and M-CDKs are functionally specialized, with substantially different substrate specificities to execute different cell cycle events 4 – 6 . A second model proposes that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall CDK activity 7 , 8 . In this model, increasing CDK activity, rather than CDK substrate specificity, orders cell cycle events 9 , 10 . Here we reconcile these two views of core cell cycle control. Using phosphoproteomic assays of in vivo CDK activity in fission yeast, we find that S-CDK and M-CDK substrate specificities are remarkably similar, showing that S-CDKs and M-CDKs are not completely specialized for S phase and mitosis alone. Normally, S-CDK cannot drive mitosis but can do so when protein phosphatase 1 is removed from the centrosome. Thus, increasing S-CDK activity in vivo is sufficient to overcome substrate specificity differences between S-CDK and M-CDK, and allows S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine is largely based on a quantitative increase in CDK activity through the cell cycle, combined with minor and surmountable qualitative differences in catalytic specialization of S-CDKs and M-CDKs. The core cell cycle is largely driven by increasing total CDK activity together with minor differences in the substrate specificity of the CDKs initiating DNA replication and mitosis.

The ribosome is a molecular machine responsible for protein synthesis and a major target for small-molecule inhibitors. Compared to the wealth of structural information available on ribosome-targeting antibiotics in bacteria, our understanding of the binding mode of ribosome inhibitors in eukaryotes is currently limited. Here we used X-ray crystallography to determine 16 high-resolution structures of 80S ribosomes from Saccharomyces cerevisiae in complexes with 12 eukaryote-specific and 4 broad-spectrum inhibitors. All inhibitors were found associated with messenger RNA and transfer RNA binding sites. In combination with kinetic experiments, the structures suggest a model for the action of cycloheximide and lactimidomycin, which explains why lactimidomycin, the larger compound, specifically targets the first elongation cycle. The study defines common principles of targeting and resistance, provides insights into translation inhibitor mode of action and reveals the structural determinants responsible for species selectivity which could guide future drug development. Whereas previous structural investigation of ribosome inhibitors has been done using the prokaryotic ribosome, this work presents X-ray crystal structures of the yeast ribosome in complex with 16 inhibitors including eukaryotic-specific inhibitors; the inhibitors all bind the mRNA or tRNA binding sites, larger molecules appear to target specifically the first elongation cycle. Mechanisms of eukaryotic ribosome inhibition As the ribosome is a common target of antibiotics, there is a wealth of structural data on the binding of the bacterial ribosome to various inhibitors. Our understanding of inhibitor binding to the larger eukaryotic ribosome is limited. Marat Yusupov and colleagues present the structure of the yeast 80S ribosome bound to 12 eukaryote-specific and 4 broad-spectrum inhibitors. On the basis of structural data and kinetic studies, the authors propose a model for the action of cycloheximide and lactimidomycin that demonstrates that the size of an inhibitor can dictate its accessibility to the ribosome and thus its mechanism of action. This new model suggests general principles for structure-based design of new antibiotics as well as therapeutics against fungal and protozoan infections, cancers and genetic disorders induced by premature stop codons.

Caspases are at the core of executing apoptosis by orchestrating cellular destruction with proteolytic cascades. Caspase-mediated proteolysis also controls diverse nonlethal cellular activities such as proliferation, differentiation, cell fate decision, and cytoskeletal reorganization. During the last decade or so, genetic studies of Drosophila have contributed to our understanding of the in vivo mechanism of the non-apoptotic cellular responses in developmental contexts. Furthermore, recent studies using C. elegans suggest that apoptotic signaling may play unexpected roles, which influence ageing and normal development at the organism level. In this review, we describe how the caspase activity is elaborately controlled during vital cellular processes at the level of subcellular localization, the duration and timing to avoid full apoptotic consequences, and also discuss the novel roles of non-apoptotic caspase signaling in adult homeostasis and physiology.

Caspases are a family of cysteine proteases widely known as the principal mediators of the apoptotic cell death response, but considerably less so as the contributors to the regulation of pathways outside cellular demise. In regards to autophagy, the modulatory roles of caspases have only recently begun to be adequately described. In contrast to apoptosis, autophagy promotes cell survival by providing energy and nutrients through the lysosomal degradation of cytoplasmic constituents. Under basal conditions autophagy and apoptosis cross-regulate each other through an elaborate network of interconnections which also includes the interplay between autophagy-related proteins (ATGs) and caspases. In this review we focus on the effects of this crosstalk at the cellular level, as we aim to concentrate the main observations from research conducted so far on the fine-tuning of autophagy by caspases. Several members of this protease-family have been found to directly interact with key ATGs involved in different tiers across the autophagic cascade. Therefore, we firstly outline the core mechanism of macroautophagy in brief. In an effort to emphasize the importance of the intricate cross-regulation of ATGs and caspases, we also present examples of autophagy’s contribution to apoptotic cell death during development.

The phenotypic and biochemical similarities between caspase-mediated apoptosis and cellular differentiation are striking. They include such diverse phenomenon as mitochondrial membrane perturbations, cytoskeletal rearrangements and DNA fragmentation. The parallels between the two disparate processes suggest some common ancestry and highlight the paradoxical nature of the death-centric view of caspases. That is, what is the driving selective pressure that sustains death-inducing proteins throughout eukaryotic evolution? Plausibly, caspase function may be rooted in a primordial non-death function, such as cell differentiation, and was co-opted for its role in programmed cell death. This review will delve into the links between caspase-mediated apoptosis and cell differentiation and examine the distinguishing features of these events. More critically, we chronicle the evolutionary origins of caspases and propose that caspases may have held an ancient role in mediating the fidelity of cell division/differentiation through its effects on proteostasis and protein quality control.

The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 Å crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded and processed by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1–8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2′-hydroxyl groups pre-organizing the backbone of nucleotides 2–8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide–target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage. Argonaute proteins are an essential part of the guide-RNA–protein complex that carries out RNA-induced gene silencing; structure–function studies of the yeast complex reveal conserved features of the eukaryotic complex, which underlie formation of the catalytically active conformation. Chance meeting for Argonaute The functional complex that carries out RNA-induced gene silencing consists of an Argonaute (Ago) protein bound to a short single-stranded guide RNA. This complex recognizes and binds a complementary messenger RNA sequence and mediates either RNA cleavage or repression of its translation. The 3.2-Ångström crystal structure of Kluyveromyces polysporus Argonaute, bound by chance to nonspecific guide RNA, has now been determined. The prokaryotic and human Ago protein structures have been solved in inactive conformations, but this yeast Ago structure is in an active conformation that reveals the mechanism of catalysis.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of synthesis of sucrose, cellulose, and several other polysaccharides in all plants. The protein is evolutionarily conserved among eukaryotes, but has little relation, aside from its catalytic reaction, to UGPases of prokaryotic origin. Using protein homology modeling strategy, 3D structures for barley, poplar, and Arabidopsis UGPases have been derived, based on recently published crystal structure of human UDP-N-acetylglucosamine pyrophosphorylase. The derived 3D structures correspond to a bowl-shaped protein with the active site at a central groove, and a C-terminal domain that includes a loop (I-loop) possibly involved in dimerization. Data on a plethora of earlier described UGPase mutants from a variety of eukaryotic organisms have been revisited, and we have, in most cases, verified the role of each mutation in enzyme catalysis/regulation/structural integrity. We have also found that one of two alternatively spliced forms of poplar UGPase has a very short I-loop, suggesting differences in oligomerization ability of the two isozymes. The derivation of the structural model for plant UGPase should serve as a useful blueprint for further function/structure studies on this protein.

Much structural information has been amassed on ATP-binding cassette (ABC) transporters, including hundreds of structures of isolated domains and an increasing array of full-length transporters. The structures capture different steps in the transport cycle and have aided in the design and interpretation of computational simulations and biophysics experiments. These data provide a maturing, although still incomplete, elucidation of the protein dynamics and mechanisms of substrate selection and transit through the transporters. We present an updated view of the classical alternating-access mechanism as it applies to eukaryotic ABC transporters, focusing on type I exporters. Our model helps frame the progress in, and remaining questions about, transporter energetics, how substrates are selected and how ATP is consumed to perform work at the molecular scale. Many human ABC transporters are associated with disease; we highlight progress in understanding their pharmacology through the lens of structural biology and describe how this knowledge suggests approaches to pharmacologically targeting these transporters.