We report the cryo-EM structure of the human 26S proteasome at an average resolution of 3.5 Å, allowing atomic modeling of 28 subunits in the core particle (CP) and 18 subunits in the regulatory particle (RP). The C-terminal residues of Rpt3 and Rpt5 subunits in the RP can be seen inserted into surface pockets formed between adjacent α subunits in the CP. Each of the six Rpt subunits contains a bound nucleotide, and the central gate of the CP α-ring is closed despite RP association. The six pore 1 loops in the Rpt ring are arranged similarly to a spiral staircase along the axial channel of substrate transport, which is constricted by the pore 2 loops. We also determined the cryo-EM structure of the human proteasome bound to the deubiquitinating enzyme USP14 at 4.35-Å resolution. Together, our structures provide a framework for mechanistic understanding of eukaryotic proteasome function.

Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1 R1074H and PHF5A Y36C . The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure-activity relationship (SAR) on the PHF5A R38C mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age.

Members of the Planctomycetes clade share many unusual features for bacteria. Their cytoplasm contains membrane-bound compartments, they lack peptidoglycan and FtsZ, they divide by polar budding, and they are capable of endocytosis. Planctomycete genomes have remained enigmatic, generally being quite large (up to 9 Mb), and on average, 55% of their predicted proteins are of unknown function. Importantly, proteins related to the unusual traits of Planctomycetes remain largely unknown. Thus, we embarked on bioinformatic analyses of these genomes in an effort to predict proteins that are likely to be involved in compartmentalization, cell division, and signal transduction. We used three complementary strategies. First, we defined the Planctomycetes core genome and subtracted genes of well-studied model organisms. Second, we analyzed the gene content and synteny of morphogenesis and cell division genes and combined both methods using a "guilt-by-association" approach. Third, we identified signal transduction systems as well as sigma factors. These analyses provide a manageable list of candidate genes for future genetic studies and provide evidence for complex signaling in the Planctomycetes akin to that observed for bacteria with complex life-styles, such as Myxococcus xanthus.

The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6-to 6.3-Å resolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function.protein degradation | proteasome | cryo-EM | structure T he eukaryotic ubiquitin-proteasome system is responsible for the degradation of polyubiquitinated proteins (1). The 26S proteasome consists of one 20S core particle (CP) and two 19S regulatory particles (RPs). The RP is divided into the lid and base assembly intermediates (1). The lid comprises nine Rpn subunits in yeast (Rpn3/5/6/7/8/9/11/12/15) and the base comprises three Rpn subunits (Rpn1/2/13) and six ATPases (Rpt1-6). Rpn10, which consists of an N-terminal von Willebrand factor A (VWA) domain and multiple C-terminal ubiquitin-interacting motifs (UIM), connects the lid and the base. Polyubiquitin (poly-Ub) chains from substrate are recognized by the RP, leading to unfolding of the substrate and its translocation into the CP, where it is degraded.The main function of the lid is to remove poly-Ub chains from the substrate (1). The released Ub chains are recycled via further cleavage into Ub monomers. Six of the nine Rpn subunits in the lid (Rpn3/5/6/7/9/12) contain a solenoid fold followed by a proteasome-CSN-eIF3 (PCI) domain of varying lengths; for Rpn8 and Rpn11, each has an Mpr1-Pad1-N-terminal (MPN) domain. Among all Rpn subunits, Rpn11 is the only deubiquitylating enzyme (DUB); it cleaves the isopeptide bond between the carboxyl terminus of Ub and the e-amino group of Lys in the substrate (2, 3). Except for Rpn15/ Sem1/Dss1, the C-terminal sequences of the other eight Rpn subunits in the lid form a helix bundle, which dictates lid assembly (4, 5).In the base, the six Rpt subunits form a hexameric ring. Powered by ATP hydrolysis, the Rpt ring is responsible for substrate unfolding and translocation of the unfolded substrate through the narrow RP central channel into the CP for degradation (6-8). The barrel-shaped CP consists of two outer α-rings and two inner β-rings, each containing seven subunits (α1-7 or β1-7). X-ray structures of the CP at atomic resolution have been reported for archaeabacteria (9), yeast (10), and mammals (11).Crystallization of the RP or the 26S proteasome is hampered by its dynamic nature. Improvement of cryo-EM technologies has ...

The 26S proteasome at the center of the ubiquitin-proteasome system (UPS) is essential for virtually all cellular processes of eukaryotes. A common misconception about the proteasome is that, once made, it remains as a static and uniform complex with spontaneous and constitutive activity for protein degradation. Recent discoveries have provided compelling evidence to support the exact opposite insomuch as the 26S proteasome undergoes dynamic and reversible phosphorylation under a variety of physiopathological conditions. In this review, we summarize the history and current understanding of proteasome phosphorylation, and advocate the idea of targeting proteasome kinases/phosphatases as a new strategy for clinical interventions of several human diseases.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-017-0382-x) contains supplementary material, which is available to authorized users.

Signal transduction is an essential process that allows bacteria to sense their complex and ever-changing environment and adapt accordingly. Three distinct major types of signal-transducing proteins (STPs) can be distinguished: one-component systems (1CSs), two-component systems (2CSs), and extracytoplasmic-function factors (ECFs). Since Actinobacteria are particularly rich in STPs, we comprehensively investigated the abundance and diversity of STPs encoded in 119 actinobacterial genomes, based on the data stored in the Microbial Signal Transduction (MiST) database. Overall, we observed an approximately linear correlation between the genome size and the total number of encoded STPs. About half of all membrane-anchored 1CSs are protein kinases. For both 1CSs and 2CSs, a detailed analysis of the domain architectures identified novel proteins that are found only in actinobacterial genomes. Many actinobacterial genomes are particularly enriched for ECFs. As a result of this study, almost 500 previously unclassified ECFs could be classified into 18 new ECF groups. This comprehensive survey demonstrates that actinobacterial genomes encode previously unknown STPs, which may represent new mechanisms of signal transduction and regulation. This information not only expands our knowledge of the diversity of bacterial signal transduction but also provides clear and testable hypotheses about their mechanisms, which can serve as starting points for experimental studies. IMPORTANCEIn the wake of the genomic era, with its enormous increase in the amount of available sequence information, the challenge has now shifted toward making sense and use of this treasure chest. Such analyses are a prerequisite to provide meaningful information that can help guide subsequent experimental efforts, such as mechanistic studies on novel signaling strategies. This work provides a comprehensive analysis of signal transduction proteins from 119 actinobacterial genomes. We identify, classify, and describe numerous novel and conserved signaling devices. Hence, our work serves as an important resource for any researcher interested in signal transduction of this important bacterial phylum, which contains organisms of ecological, biotechnological, and medical relevance. Bacterial survival critically depends on the ability to swiftly respond to environmental changes. To efficiently monitor the surrounding environment, microbial genomes encode numerous and highly diverse proteins that can sense a given extracellular stimulus, transmit the signal to the cytoplasm, and elicit a proper response. These signal-transducing proteins (STPs) can be divided into three major groups: one-component systems (1CSs), two-component systems (2CSs), and extracytoplasmic-function factors (ECFs). The vast majority of STPs in bacteria are 1CSs. These systems are composed of a single protein that contains an input domain, which senses the stimulus, and an output domain, which elicits the response by binding nucleic acids, modifying proteins, or performing an enzymati...

Occult hepatitis B (OHB) infection has been reported to play an important role in the development of hepatocellular carcinoma (HCC). In this systematic review, a significantly higher prevalence of OHB was observed in patients with HCC in the presence or absence of HCV infection when compared with control populations without HCC. Correspondingly, among adequately designed prospective studies, the cumulative probability of developing HCC was significantly greater among patients with OHB than among HBV DNA-negative patients in the presence or absence of HCV infection. Study design, inclusion criteria, treatment options, methodology and potential confounding variables were evaluated, and immunopathogenic mechanisms that could be involved in OHB as a risk factor in HCC were reviewed. From this analysis, we conclude that although OHB is an independent risk factor in HCC development in anti-HCV-negative patients, a synergistic or additive role in the occurrence of HCC in HCV-coinfected patients is more problematic due to the HCC risk attributable to HCV alone, especially in patients with advanced fibrosis and cirrhosis.

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