PemB, a type III secretion effector in Pseudomonas aeruginosa, affects Caenorhabditis elegans life span.

Pseudomonas aeruginosa is one of the leading nosocomial opportunistic pathogens causing acute and chronic infections. Among its main virulent factors is the Type III secretion system (T3SS) which enhances disease severity by delivering effectors to the host in a highly regulated manner. Despite its importance for virulence, only six T3SS-dependent effectors have been discovered so far. Previously, we identified two new potential effectors using a machine-learning algorithm approach. Here we demonstrate that one of these effectors, PemB, is indeed virulent. Using a live Caenorhabditis elegans infection model, we demonstrate this effector damages the integrity of the intestine barrier leading to the death of the host. Implementing a high-throughput assay using Saccharomyces cerevisiae, we identified several candidate proteins that interact with PemB. One of them, EFT1, has an ortholog in C. elegans (eef-2) and is also an essential gene and a well-known target utilized by different pathogens to induce toxicity to the worm. Accordingly, we found that by silencing the eef-2 gene in C. elegans, PemB could no longer induce its toxic effect. The current study further uncovers the complex machinery assisting P. aeruginosa virulence and may provide novel insight how to manage infection associated with this hard-to-treat pathogen.

A pipeline for identifying guide RNA sequences that promote RNA editing of nonsense mutations that cause inherited retinal diseases.

Adenosine deaminases acting on RNA (ADARs) are endogenous enzymes catalyzing the deamination of adenosines to inosines, which are then read as guanosines during translation. This ability to recode makes ADAR an attractive therapeutic tool to edit genetic mutations and reprogram genetic information at the mRNA level. Using the endogenous ADARs and guiding them to a selected target has promising therapeutic potential. Indeed, different studies have reported several site-directed RNA-editing approaches for making targeted base changes in RNA molecules. The basic strategy has been to use guide RNAs (gRNAs) that hybridize and form a double-stranded RNA (dsRNA) structure with the desired RNA target because of ADAR activity in regions of dsRNA formation. Here we report on a novel pipeline for identifying disease-causing variants as candidates for RNA editing, using a yeast-based screening system to select efficient gRNAs for editing of nonsense mutations, and test them in a human cell line reporter system. We have used this pipeline to modify the sequence of transcripts carrying nonsense mutations that cause inherited retinal diseases in the FAM161A, KIZ, TRPM1, and USH2A genes. Our approach can serve as a basis for gene therapy intervention in knockin mouse models and ultimately in human patients.

DNA and RNA base editors can correct the majority of pathogenic single nucleotide variants.

The majority of human genetic diseases are caused by single nucleotide variants (SNVs) in the genome sequence. Excitingly, new genomic techniques known as base editing have opened efficient pathways to correct erroneous nucleotides. Due to reliance on deaminases, which have the capability to convert A to I(G) and C to U, the direct applicability of base editing might seem constrained in terms of the range of mutations that can be reverted. In this evaluation, we assess the potential of DNA and RNA base editing methods for treating human genetic diseases. Our findings indicate that 62% of pathogenic SNVs found within genes can be amended by base editing; 30% are G>A and T>C SNVs that can be corrected by DNA base editing, and most of them by RNA base editing as well, and 29% are C>T and A>G SNVs that can be corrected by DNA base editing directed to the complementary strand. For each, we also present several factors that affect applicability such as bystander and off-target occurrences. For cases where editing the mismatched nucleotide is not feasible, we introduce an approach that calculates the optimal substitution of the deleterious amino acid with a new amino acid, further expanding the scope of applicability. As personalized therapy is rapidly advancing, our demonstration that most SNVs can be treated by base editing is of high importance. The data provided will serve as a comprehensive resource for those seeking to design therapeutic base editors and study their potential in curing genetic diseases.

Dissecting the basis for differential substrate specificity of ADAR1 and ADAR2.

Millions of adenosines are deaminated throughout the transcriptome by ADAR1 and/or ADAR2 at varying levels, raising the question of what are the determinants guiding substrate specificity and how these differ between the two enzymes. We monitor how secondary structure modulates ADAR2 vs ADAR1 substrate selectivity, on the basis of systematic probing of thousands of synthetic sequences transfected into cell lines expressing exclusively ADAR1 or ADAR2. Both enzymes induce symmetric, strand-specific editing, yet with distinct offsets with respect to structural disruptions: -26 nt for ADAR2 and -35 nt for ADAR1. We unravel the basis for these differences in offsets through mutants, domain-swaps, and ADAR homologs, and find it to be encoded by the differential RNA binding domain (RBD) architecture. Finally, we demonstrate that this offset-enhanced editing can allow an improved design of ADAR2-recruiting therapeutics, with proof-of-concept experiments demonstrating increased on-target and potentially decreased off-target editing.

Identification of exceptionally potent adenosine deaminases RNA editors from high body temperature organisms.

The most abundant form of RNA editing in metazoa is the deamination of adenosines into inosines (A-to-I), catalyzed by ADAR enzymes. Inosines are read as guanosines by the translation machinery, and thus A-to-I may lead to protein recoding. The ability of ADARs to recode at the mRNA level makes them attractive therapeutic tools. Several approaches for Site-Directed RNA Editing (SDRE) are currently under development. A major challenge in this field is achieving high on-target editing efficiency, and thus it is of much interest to identify highly potent ADARs. To address this, we used the baker yeast Saccharomyces cerevisiae as an editing-naïve system. We exogenously expressed a range of heterologous ADARs and identified the hummingbird and primarily mallard-duck ADARs, which evolved at 40-42°C, as two exceptionally potent editors. ADARs bind to double-stranded RNA structures (dsRNAs), which in turn are temperature sensitive. Our results indicate that species evolved to live with higher core body temperatures have developed ADAR enzymes that target weaker dsRNA structures and would therefore be more effective than other ADARs. Further studies may use this approach to isolate additional ADARs with an editing profile of choice to meet specific requirements, thus broadening the applicability of SDRE.

Reversal of histone H2B mono-ubiquitination is required for replication stress recovery.

Mono-ubiquitination of histone H2B (H2B-Ub1) is a conserved modification that plays central role in regulating numerous biological processes including the DNA damage response, gene transcription, and DNA replication. Previous studies have revealed that H2B-Ub1 promotes recovery from replication stress by mediating Rad53 phosphorylation (Rad53-P), and activation of the intra-S replication checkpoint, in order to limit fork progression, and associated DNA damage. Since such mono-ubiquitination is a reversible process, we examined the role of H2B-Ub1 deubiquitination during replication stress. Using an experimental system in yeast which mimics H2B-Ub1 accumulation, we show that cells become sensitive to the replication stress induced by HU. This stress response was accompanied by Rad53-P accumulation, and delayed recovery from intra-S checkpoint arrest. Furthermore, we show that similar effects were recapitulated by the accumulation of endogenous H2B-Ub1, induced by the co-inactivation of the deubiquitinating enzyme, Ubp10, and Spt16, a FACT histone chaperone family member. While it has been well established that H2B mono-ubiquitination plays an essential role in recovering from replication stress, our data reveal that H2B-Ub1 deubiquitination is also essential for this process.

Inherited retinal diseases: Linking genes, disease-causing variants, and relevant therapeutic modalities.

Inherited retinal diseases (IRDs) are a clinically complex and heterogenous group of visual impairment phenotypes caused by pathogenic variants in at least 277 nuclear and mitochondrial genes, affecting different retinal regions, and depleting the vision of affected individuals. Genes that cause IRDs when mutated are unique by possessing differing genotype-phenotype correlations, varying inheritance patterns, hypomorphic alleles, and modifier genes thus complicating genetic interpretation. Next-generation sequencing has greatly advanced the identification of novel IRD-related genes and pathogenic variants in the last decade. For this review, we performed an in-depth literature search which allowed for compilation of the Global Retinal Inherited Disease (GRID) dataset containing 4,798 discrete variants and 17,299 alleles published in 31 papers, showing a wide range of frequencies and complexities among the 194 genes reported in GRID, with 65% of pathogenic variants being unique to a single individual. A better understanding of IRD-related gene distribution, gene complexity, and variant types allow for improved genetic testing and therapies. Current genetic therapeutic methods are also quite diverse and rely on variant identification, and range from whole gene replacement to single nucleotide editing at the DNA or RNA levels. IRDs and their suitable therapies thus require a range of effective disease modelling in human cells, granting insight into disease mechanisms and testing of possible treatments. This review summarizes genetic and therapeutic modalities of IRDs, provides new analyses of IRD-related genes (GRID and complexity scores), and provides information to match genetic-based therapies such as gene-specific and variant-specific therapies to the appropriate individuals.

Inactive Proteasomes Routed to Autophagic Turnover Are Confined within the Soluble Fraction of the Cell.

Previous studies demonstrated that dysfunctional yeast proteasomes accumulate in the insoluble protein deposit (IPOD), described as the final deposition site for amyloidogenic insoluble proteins and that this compartment also mediates proteasome ubiquitination, a prerequisite for their targeted autophagy (proteaphagy). Here, we examined the solubility state of proteasomes subjected to autophagy as a result of their inactivation, or under nutrient starvation. In both cases, only soluble proteasomes could serve as a substrate to autophagy, suggesting a modified model whereby substrates for proteaphagy are dysfunctional proteasomes in their near-native soluble state, and not as previously believed, those sequestered at the IPOD. Furthermore, the insoluble fraction accumulating in the IPOD represents an alternative pathway, enabling the removal of inactive proteasomes that escaped proteaphagy when the system became saturated. Altogether, we suggest that the relocalization of proteasomes to soluble aggregates represents a general stage of proteasome recycling through autophagy.

Regulation of the anaphase promoting complex/cyclosome by the degradation of its unassembled catalytic subunit, Apc11.

One of the challenges encountered by the protein quality control machinery is the need to ensure that members of multiprotein complexes are available in the correct proportions. In this study, we demonstrate that the ubiquitin proteasome system (UPS) mediates the degradation of Apc11, the catalytic core subunit of the anaphase promoting complex/cyclosome (APC/C). In vitro studies have shown that Apc11, together with its E2 enzyme, is sufficient to ubiquitinate substrates independently of the APC/C. Here, we establish that this can occur in living yeast cells. We show that the tight controls regulating the function of the fully assembled APC/C can be circumvented when its substrates are ubiquitinated by the excess levels of Apc11 independently of the assembled complex. We thus suggest that the UPS-mediated degradation of Apc11 is an overlooked mechanism ensuring that proper function of the APC/C is limited to suitably delimited holoenzymes and that an imbalance in protein expression may result in detrimental gain-of-function activity, rather than merely the disruption of protein complex stoichiometry.-Volpe, M., Levinton, N., Rosenstein, N., Prag, G., Ben-Aroya, S. Regulation of the anaphase promoting complex/cyclosome by the degradation of its unassembled catalytic subunit, Apc11.

Spatial Organization of Proteasome Aggregates in the Regulation of Proteasome Homeostasis.

Misfolded proteins and insoluble aggregates are continuously produced in the cell and can result in severe stress that threatens cellular fitness and viability if not managed effectively. Accordingly, organisms have evolved several protective protein quality control (PQC) machineries to address these threats. In eukaryotes, the ubiquitin-proteasome system (UPS) plays a vital role in the disposal of intracellular misfolded, damaged, or unneeded proteins. Although ubiquitin-mediated proteasomal degradation of many proteins plays a key role in the PQC system, cells must also dispose of the proteasomes themselves when their subunits are assembled improperly, or when they dysfunction under various conditions, e.g., as a result of genomic mutations, diverse stresses, or treatment with proteasome inhibitors. Here, we review recent studies that identified the regulatory pathways that mediate proteasomes sorting under various stress conditions, and the elimination of its dysfunctional subunits. Following inactivation of the 26S proteasome, UPS-mediated degradation of its own misassembled subunits is the favored disposal pathway. However, the cytosolic cell-compartment-specific aggregase, Hsp42 mediates an alternative pathway, the accumulation of these subunits in cytoprotective compartments, where they become extensively modified with ubiquitin, and are directed by ubiquitin receptors for autophagic clearance (proteaphagy). We also discuss the sorting mechanisms that the cell uses under nitrogen stress, and to distinguish between dysfunctional proteasome aggregates and proteasome storage granules (PSGs), reversible assemblies of membrane-free cytoplasmic condensates that form in yeast upon carbon starvation and help protect proteasomes from autophagic degradation. Regulated proteasome subunit homeostasis is thus controlled through cellular probing of the level of proteasome assembly, and the interplay between UPS-mediated degradation or sorting of misfolded proteins into distinct cellular compartments.

Fe-S cluster coordination of the chromokinesin KIF4A alters its subcellular localization during mitosis.

Fe-S clusters act as co-factors of proteins with diverse functions, for example, in DNA repair. Downregulation of the cytosolic iron-sulfur protein assembly (CIA) machinery promotes genomic instability through the inactivation of multiple DNA repair pathways. Furthermore, CIA deficiencies are associated with so far unexplained mitotic defects. Here, we show that CIA2B (also known as FAM96B) and MMS19, constituents of the CIA targeting complex involved in facilitating Fe-S cluster insertion into cytosolic and nuclear target proteins, colocalize with components of the mitotic machinery. Downregulation of CIA2B and MMS19 impairs the mitotic cycle. We identify the chromokinesin KIF4A as a mitotic component involved in these effects. KIF4A binds a Fe-S cluster in vitro through its conserved cysteine-rich domain. We demonstrate in vivo that this domain is required for the mitosis-related KIF4A localization and for the mitotic defects associated with KIF4A knockout. KIF4A is the first identified mitotic component carrying such a post-translational modification. These findings suggest that the lack of Fe-S clusters in KIF4A upon downregulation of the CIA targeting complex contributes to the mitotic defects.

A-to-I RNA Editing: An Overlooked Source of Cancer Mutations.

RNA editing is a source of transcriptomic diversity, mainly in non-coding regions, and is found to be altered in cancer. In this issue of Cancer Cell, Peng et al. show that RNA editing events are manifested at the proteomic levels and are a source of cancer protein heterogeneity.

Chemical inhibitors of Candida albicans hyphal morphogenesis target endocytosis.

Candida albicans is an opportunistic pathogen, typically found as a benign commensal yeast living on skin and mucosa, but poised to invade injured tissue to cause local infections. In debilitated and immunocompromised individuals, C. albicans may spread to cause life-threatening systemic infections. Upon contact with serum and at body temperature, C. albicans performs a regulated switch to filamentous morphology, characterized by emergence of a germ tube from the yeast cell followed by mold-like growth of branching hyphae. The ability to switch between growth morphologies is an important virulence factor of C. albicans. To identify compounds able to inhibit hyphal morphogenesis, we screened libraries of existing drugs for inhibition of the hyphal switch under stringent conditions. Several compounds that specifically inhibited hyphal morphogenesis were identified. Chemogenomic analysis suggested an interaction with the endocytic pathway, which was confirmed by direct measurement of fluid-phase endocytosis in the presence of these compounds. These results suggest that the activity of the endocytic pathway, which is known to be particularly important for hyphal growth, represents an effective target for hyphae-inhibiting drugs.

A New Method, "Reverse Yeast Two-Hybrid Array" (RYTHA), Identifies Mutants that Dissociate the Physical Interaction Between Elg1 and Slx5.

The vast majority of processes within the cell are carried out by proteins working in conjunction. The Yeast Two-Hybrid (Y2H) methodology allows the detection of physical interactions between any two interacting proteins. Here, we describe a novel systematic genetic methodology, "Reverse Yeast Two-Hybrid Array" (RYTHA), that allows the identification of proteins required for modulating the physical interaction between two given proteins. Our assay starts with a yeast strain in which the physical interaction of interest can be detected by growth on media lacking histidine, in the context of the Y2H methodology. By combining the synthetic genetic array technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify trans-acting mutations that disrupt the physical interaction of interest. We apply this novel method in a screen for mutants that disrupt the interaction between the N-terminus of Elg1 and the Slx5 protein. Elg1 is part of an alternative replication factor C-like complex that unloads PCNA during DNA replication and repair. Slx5 forms, together with Slx8, a SUMO-targeted ubiquitin ligase (STUbL) believed to send proteins to degradation. Our results show that the interaction requires both the STUbL activity and the PCNA unloading by Elg1, and identify topoisomerase I DNA-protein cross-links as a major factor in separating the two activities. Thus, we demonstrate that RYTHA can be applied to gain insights about particular pathways in yeast, by uncovering the connection between the proteasomal ubiquitin-dependent degradation pathway, DNA replication, and repair machinery, which can be separated by the topoisomerase-mediated cross-links to DNA.

A bacterial genetic selection system for ubiquitylation cascade discovery.

About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.

Structure of ubiquitylated-Rpn10 provides insight into its autoregulation mechanism.

Ubiquitin receptors decode ubiquitin signals into many cellular responses. Ubiquitin receptors also undergo coupled monoubiquitylation, and rapid deubiquitylation has hampered the characterization of the ubiquitylated state. Using bacteria that express a ubiquitylation apparatus, we purified and determined the crystal structure of the proteasomal ubiquitin-receptor Rpn10 in its ubiquitylated state. The structure shows a novel ubiquitin-binding patch that directs K84 ubiquitylation. Superimposition of ubiquitylated-Rpn10 onto electron-microscopy models of proteasomes indicates that the Rpn10-conjugated ubiquitin clashes with Rpn9, suggesting that ubiquitylation might be involved in releasing Rpn10 from the proteasome. Indeed, ubiquitylation on immobilized proteasomes dissociates the modified Rpn10 from the complex, while unmodified Rpn10 mainly remains associated. In vivo experiments indicate that contrary to wild type, Rpn10-K84R is stably associated with the proteasomal subunit Rpn9. Similarly Rpn10, but not ubiquitylated-Rpn10, binds Rpn9 in vitro. Thus we suggest that ubiquitylation functions to dissociate modified ubiquitin receptors from their targets, a function that promotes cyclic activity of ubiquitin receptors.

Proteasome storage granules are transiently associated with the insoluble protein deposit in Saccharomyces cerevisiae.

Proteasome storage granules (PSGs) are created in yeast as part of an extensive and programmed reorganization of proteins into reversible assemblies upon carbon source depletion. Here, we demonstrate that cells distinguish dysfunctional proteasomes from PSGs on the cytosolic insoluble protein deposit (IPOD). Furthermore, we provide evidence that this is a general mechanism for the reorganization of additional proteins into reversible assemblies. Our study expands the roles of the IPOD, which might serve not only as the specific depository for amyloidogenic and misfolded proteins, but also as a potential hub from which proteins are directed to distinct cellular compartments. These findings therefore provide a framework for understanding how cells discriminate between intact and abnormal proteins under stress conditions to ensure that only structurally 'correct' proteins are deployed.

An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes.

Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes.

The protein quality control machinery regulates its misassembled proteasome subunits.

Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with various aggregation diseases. In eukaryotes, the ubiquitin proteasome system (UPS) plays a vital role in protein quality control (PQC), by selectively targeting misfolded proteins for degradation. While the assembly of the proteasome can be naturally impaired by many factors, the regulatory pathways that mediate the sorting and elimination of misassembled proteasomal subunits are poorly understood. Here, we reveal how the dysfunctional proteasome is controlled by the PQC machinery. We found that among the multilayered quality control mechanisms, UPS mediated degradation of its own misassembled subunits is the favored pathway. We also demonstrated that the Hsp42 chaperone mediates an alternative pathway, the accumulation of these subunits in cytoprotective compartments. Thus, we show that proteasome homeostasis is controlled through probing the level of proteasome assembly, and the interplay between UPS mediated degradation or their sorting into distinct cellular compartments.

Novel type III effectors in Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes chronic and acute infections in immunocompromised patients. Most P. aeruginosa strains encode an active type III secretion system (T3SS), utilized by the bacteria to deliver effector proteins from the bacterial cell directly into the cytoplasm of the host cell. Four T3SS effectors have been discovered and extensively studied in P. aeruginosa: ExoT, ExoS, ExoU, and ExoY. This is especially intriguing in light of P. aeruginosa's ability to infect a wide range of hosts. We therefore hypothesized that additional T3SS effectors that have not yet been discovered are encoded in the genome of P. aeruginosa. Here, we applied a machine learning classification algorithm to identify novel P. aeruginosa effectors. In this approach, various types of data are integrated to differentiate effectors from the rest of the open reading frames of the bacterial genome. Due to the lack of a sufficient learning set of positive effectors, our machine learning algorithm integrated genomic information from another Pseudomonas species and utilized dozens of features accounting for various aspects of the effector coding genes and their products. Twelve top-ranking predictions were experimentally tested for T3SS-specific translocation, leading to the discovery of two novel T3SS effectors. We demonstrate that these effectors are not part of the injection structural complex and report initial efforts toward their characterization. IMPORTANCE: Pseudomonas aeruginosa uses a type III secretion system (T3SS) to secrete toxic proteins, termed effectors, directly into the cytoplasm of the host cell. The activation of this secretion system is correlated with disease severity and patient death. Compared with many other T3SS-utilizing pathogenic bacteria, P. aeruginosa has a fairly limited arsenal of effectors that have been identified. This is in sharp contrast with the wide range of hosts that this bacterium can infect. The discovery of two novel effectors described here is an important step toward better understanding of the virulence and host evasion mechanisms adopted by this versatile pathogen and may provide novel approaches to treat P. aeruginosa infections.