J. Appl. Cryst.の発刊に際して

50 Years of Image Analysis

Ten measures of experimental electron-density-map quality are examined and the skewness of electron density is found to be the best indicator of actual map quality. A Bayesian approach to estimating map quality is developed and used in the PHENIX AutoSol wizard to make decisions during automated structure solution.

Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX autosol wizard

Protein ubiquitination is one of the most powerful posttranslational modifications of proteins, as it regulates a plethora of cellular processes in distinct manners. Simple monoubiquitination events coexist with more complex forms of polyubiquitination, the latter featuring many different chain architectures. Ubiquitin can be subjected to further posttranslational modifications (e.g., phosphorylation and acetylation) and can also be part of mixed polymers with ubiquitin-like modifiers such as SUMO (small ubiquitin-related modifier) or NEDD8 (neural precursor cell expressed, developmentally downregulated 8). Together, cellular ubiquitination events form a sophisticated and versatile ubiquitin code. Deubiquitinases (DUBs) reverse ubiquitin signals with equally high sophistication. In this review, we conceptualize the many layers of specificity that DUBs encompass to control the ubiquitin code and discuss examples in which DUB specificity has been understood at the molecular level. We further discuss the many mechanisms of DUB regulation with a focus on those that modulate catalytic activity. Our review provides a framework to tackle lingering questions in DUB biology.

Mechanisms of Deubiquitinase Specificity and Regulation

As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.

Structure and Function of the 26S Proteasome.

Wolbachia are obligate intracellular bacteria1 that infect arthropods, including approximately two-thirds of insect species2. Wolbachia manipulate insect reproduction by enhancing their inheritance through the female germline. The most common alteration is cytoplasmic incompatibility (CI)3-5, where eggs from uninfected females fail to develop when fertilized by sperm from Wolbachia-infected males. By contrast, if female and male partners are both infected, embryos are viable. CI is a gene-drive mechanism impacting population structure6 and causing reproductive isolation7, but its molecular mechanism has remained unknown. We show that a Wolbachia deubiquitylating enzyme (DUB) induces CI. The CI-inducing DUB, CidB, cleaves ubiquitin from substrates and is encoded in a two-gene operon, and the other protein, CidA, binds CidB. Binding is strongest between cognate partners in cidA-cidB homologues. In transgenic Drosophila, the cidA-cidB operon mimics CI when sperm introduce it into eggs, and a catalytically inactive DUB does not induce sterility. Toxicity is recapitulated in yeast by CidB alone; this requires DUB activity but is rescued by coexpressed CidA. A paralogous operon involves a putative nuclease (CinB) rather than a DUB. Analogous binding, toxicity and rescue in yeast were observed. These results identify a CI mechanism involving interacting proteins that are secreted into germline cells by Wolbachia, and suggest new methods for insect control.

/pdf/a-wolbachia-deubiquitylating-enzyme-induces-cytoplasmic-4cpb4p893y.pdf

A Wolbachia Deubiquitylating Enzyme Induces Cytoplasmic Incompatibility

Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families.

/pdf/substrate-specificity-of-the-ubiquitin-and-ubl-proteases-2iuom4ennf.pdf

Substrate specificity of the ubiquitin and Ubl proteases.

Wolbachia are endosymbiotic bacteria that infect nearly half of all arthropod species. This pandemic is due in part to their ability to increase their transmission through the female germline, most commonly by a mechanism called cytoplasmic incompatibility (CI). The Wolbachia cid operon, encoding 2 proteins, CidA and CidB, the latter a deubiquitylating enzyme (DUB), recapitulates CI in transgenic Drosophila melanogaster However, some CI-inducing Wolbachia strains lack a DUB-encoding cid operon; it was therefore proposed that the related cin operon codes for an alternative CI system. Here we show that the Wolbachia cin operon encodes a nuclease, CinB, and a second protein, CinA, that tightly binds CinB. Recombinant CinB has nuclease activity against both single-stranded and double-stranded DNA but not RNA under the conditions tested. Expression of the cin operon in transgenic male flies induces male sterility and embryonic defects typical of CI. Importantly, transgenic CinA can rescue defects in egg-hatch rates when expressed in females. Expression of CinA also rescues CinB-induced growth defects in yeast. CinB has 2 PD-(D/E)xK nuclease domains, and both are required for nuclease activity and for toxicity in yeast and flies. Our data suggest a distinct mechanism for CI involving a nuclease toxin and highlight the central role of toxin-antidote operons in Wolbachia-induced cytoplasmic incompatibility.

A Wolbachia nuclease and its binding partner provide a distinct mechanism for cytoplasmic incompatibility.

AMSH, a conserved zinc metallo deubiquitinase, controls downregulation and degradation of cell-surface receptors mediated by the endosomal sorting complexes required for transport (ESCRT) machinery. It displays high specificity toward the Lys63-linked polyubiquitin chain, which is used as a signal for ESCRT-mediated endosomal–lysosomal sorting of receptors. Herein, we report the crystal structures of the catalytic domain of AMSH orthologue Sst2 from fission yeast, its ubiquitin (product)-bound form, and its Lys63-linked diubiquitin (substrate)-bound form at 1.45, 1.7, and 2.3 A, respectively. The structures reveal that the P-side product fragment maintains nearly all the contacts with the enzyme as seen with the P portion (distal ubiquitin) of the Lys63-linked diubiquitin substrate, with additional coordination of the Gly76 carboxylate group of the product with the active-site Zn2+. One of the product-bound structures described herein is the result of an attempt to cocrystallize the diubiquitin substrate ...

http://pubs.acs.org/doi/pdf/10.1021/bi5003162

Insights into the Mechanism of Deubiquitination by JAMM Deubiquitinases from Cocrystal Structures of the Enzyme with the Substrate and Product.

Polarization-resolved second-harmonic generation (PR-SHG) microscopy is described and applied to identify the presence of multiple crystallographic domains within protein-crystal conglomerates, which was confirmed by synchrotron X-ray diffraction. Principal component analysis (PCA) of PR-SHG images resulted in principal component 2 (PC2) images with areas of contrasting negative and positive values for conglomerated crystals and PC2 images exhibiting uniformly positive or uniformly negative values for single crystals. Qualitative assessment of PC2 images allowed the identification of domains of different internal ordering within protein-crystal samples as well as differentiation between multi-domain conglomerated crystals and single crystals. PR-SHG assessments of crystalline domains were in good agreement with spatially resolved synchrotron X-ray diffraction measurements. These results have implications for improving the productive throughput of protein structure determination through early identification of multi-domain crystals.

/pdf/polarization-resolved-second-harmonic-generation-microscopy-vzp2b7kmo6.pdf

Judith A. Ronau

Papers

A Wolbachia Deubiquitylating Enzyme Induces Cytoplasmic Incompatibility

Substrate specificity of the ubiquitin and Ubl proteases.

A Wolbachia nuclease and its binding partner provide a distinct mechanism for cytoplasmic incompatibility.

Insights into the Mechanism of Deubiquitination by JAMM Deubiquitinases from Cocrystal Structures of the Enzyme with the Substrate and Product.

Polarization-resolved second-harmonic generation microscopy as a method to visualize protein-crystal domains.