LC Domain-Mediated Coalescence Is Essential for Otu Enzymatic Activity to Extend Drosophila Lifespan.

In eukaryotic cells, RNA-binding proteins (RBPs) interact with RNAs to form ribonucleoprotein complexes (RNA granules) that have long been thought to regulate RNA fate or activity. Emerging evidence suggests that some RBPs not only bind RNA but also possess enzymatic activity related to ubiquitin regulation, raising important questions of whether these RBP-formed RNA granules regulate ubiquitin signaling and related biological functions. Here, we show that Drosophila Otu binds RNAs and coalesces to membrane-less biomolecular condensates via its intrinsically disordered low-complexity domain, and coalescence represents a functional state for Otu exerting deubiquitinase activity. Notably, coalescence-mediated enzymatic activity of Otu is positively regulated by its bound RNAs and co-partner Bam. Further genetic analysis reveals that the Otu/Bam deubiquitinase complex and dTraf6 constitute a feedback loop to maintain intestinal immune homeostasis during aging, thereby controlling longevity. Thus, regulated biomolecular condensates may represent a mechanism that controls dynamic enzymatic activities and related biological processes.

Towards a unified medical microbiome ecology of the OMU for metagenomes and the OTU for microbes.

BACKGROUND: Metagenomic sequencing technologies offered unprecedented opportunities and also challenges to microbiology and microbial ecology particularly. The technology has revolutionized the studies of microbes and enabled the high-profile human microbiome and earth microbiome projects. The terminology-change from microbes to microbiomes signals that our capability to count and classify microbes (microbiomes) has achieved the same or similar level as we can for the biomes (macrobiomes) of plants and animals (macrobes). While the traditional investigations of macrobiomes have usually been conducted through naturalists' (Linnaeus & Darwin) naked eyes, and aerial and satellite images (remote-sensing), the large-scale investigations of microbiomes have been made possible by DNA-sequencing-based metagenomic technologies. Two major types of metagenomic sequencing technologies-amplicon sequencing and whole-genome (shotgun sequencing)-respectively generate two contrastingly different categories of metagenomic reads (data)-OTU (operational taxonomic unit) tables representing microorganisms and OMU (operational metagenomic unit), a new term coined in this article to represent various cluster units of metagenomic genes. RESULTS: The ecological science of microbiomes based on the OTU representing microbes has been unified with the classic ecology of macrobes (macrobiomes), but the unification based on OMU representing metagenomes has been rather limited. In a previous series of studies, we have demonstrated the applications of several classic ecological theories (diversity, composition, heterogeneity, and biogeography) to the studies of metagenomes. Here I push the envelope for the unification of OTU and OMU again by demonstrating the applications of metacommunity assembly and ecological networks to the metagenomes of human gut microbiomes. Specifically, the neutral theory of biodiversity (Sloan's near neutral model), Ning et al.stochasticity framework, core-periphery network, high-salience skeleton network, special trio-motif, and positive-to-negative ratio are applied to analyze the OMU tables from whole-genome sequencing technologies, and demonstrated with seven human gut metagenome datasets from the human microbiome project. CONCLUSIONS: All of the ecological theories demonstrated previously and in this article, including diversity, composition, heterogeneity, stochasticity, and complex network analyses, are equally applicable to OMU metagenomic analyses, just as to OTU analyses. Consequently, I strongly advocate the unification of OTU/OMU (microbiomes) with classic ecology of plants and animals (macrobiomes) in the context of medical ecology.

Regulation of Atg8 membrane deconjugation by cysteine proteases in the malaria parasite Plasmodium berghei.

During macroautophagy, the Atg8 protein is conjugated to phosphatidylethanolamine (PE) in autophagic membranes. In Apicomplexan parasites, two cysteine proteases, Atg4 and ovarian tumor unit (Otu), have been identified to delipidate Atg8 to release this protein from membranes. Here, we investigated the role of cysteine proteases in Atg8 conjugation and deconjugation and found that the Plasmodium parasite consists of both activities. We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 and Otu showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling. These findings highlight a role for the Atg8 deconjugation pathway in organelle biogenesis and maintenance of the homeostatic cellular balance.

Insight into the microbial community of denitrification process using different solid carbon sources: Not only bacteria.

There is a lack of understanding about the bacterial, fungal and archaeal communities' composition of solid-phase denitrification (SPD) systems. We investigated four SPD systems with different carbon sources by analyzing microbial gene sequences based on operational taxonomic unit (OTU) and amplicon sequence variant (ASV). The results showed that the corncob-polyvinyl alcohol sodium alginate-polycaprolactone (CPSP, 0.86±0.04 mg NO3--N/(g·day)) and corncob (0.85±0.06 mg NO3--N/(g·day)) had better denitrification efficiency than polycaprolactone (PCL, 0.29±0.11 mg NO3--N/(g·day)) and polyvinyl alcohol-sodium alginate (PVA-SA, 0.24±0.07 mg NO3--N/(g·day)). The bacterial, fungal and archaeal microbial composition was significantly different among carbon source types such as Proteobacteria in PCL (OTU: 83.72%, ASV: 82.49%) and Rozellomycota in PVA-SA (OTU: 71.99%, ASV: 81.30%). ASV methods can read more microbial units than that of OTU and exhibit higher alpha diversity and classify some species that had not been identified by OTU such as Nanoarchaeota phylum, unclassified_ f_ Xanthobacteraceae genus, etc., indicating ASV may be more conducive to understand SPD microbial communities. The co-occurring network showed some correlation between the bacteria fungi and archaea species, indicating different species may collaborate in SPD systems. Similar KEGG function prediction results were obtained in two bioinformatic methods generally and some fungi and archaea functions should not be ignored in SPD systems. These results may be beneficial for understanding microbial communities in SPD systems.

ERK/RSK-mediated phosphorylation of Y-box binding protein-1 aggravates diabetic cardiomyopathy by suppressing its interaction with deubiquitinase OTUB1.

Diabetic cardiomyopathy (DCM) is a major complication of diabetes, but its underlying mechanisms still remain unclear. The multifunctional protein Y-box binding protein-1 (YB-1) plays an important role in cardiac pathogenesis by regulating cardiac apoptosis, cardiac fibrosis, and pathological remodeling, whereas its role in chronic DCM requires further investigation. Here, we report that the phosphorylation of YB-1 at serine102 (S102) was markedly elevated in streptozotocin-induced diabetic mouse hearts and in high glucose-treated cardiomyocytes, whereas total YB-1 protein levels were significantly reduced. Coimmunoprecipitation experiments showed that YB-1 interacts with the deubiquitinase otubain-1, but hyperglycemia-induced phosphorylation of YB-1 at S102 diminished this homeostatic interaction, resulting in ubiquitination and degradation of YB-1. Mechanistically, the high glucose-induced phosphorylation of YB-1 at S102 is dependent on the upstream extracellular signal-regulated kinase (ERK)/Ras/mitogen-activated protein kinase (p90 ribosomal S6 kinase [RSK]) signaling pathway. Accordingly, pharmacological inhibition of the ERK pathway using the upstream kinase inhibitor U0126 ameliorated features of DCM compared with vehicle-treated diabetic mice. We demonstrate that ERK inhibition with U0126 also suppressed the phosphorylation of the downstream RSK and YB-1 (S102), which stabilized the interaction between YB-1 and otubain-1 and thereby preserved YB-1 protein expression in diabetic hearts. Taken together, we propose that targeting the ERK/RSK/YB-1 pathway could be a potential therapeutic approach for treating DCM.

An indispensable role of TAZ in anoikis resistance promoted by OTUB1 deubiquitinating enzyme in basal-like triple-negative breast cancer cells.

Aggressive cancers, such as triple-negative breast cancer (TNBC), are mostly fatal because of their potential to metastasize to distant organs. Cancer cells acquire various abilities to metastasize, including resistance to anoikis, an apoptotic cell death induced by loss of anchorage to the extracellular matrix. Transcriptional coactivator with PDZ binding motif (TAZ) and Yes-associated protein (YAP), the downstream effectors of the Hippo pathway, regulate cell- and tissue-level architectures by responding to mechanical microenvironments of cells, including the cell-extracellular matrix interaction. The Hippo pathway is frequently disrupted in cancer cells, and TAZ and YAP are irrelevantly activated, potentially resulting in anchorage-independent survival/proliferation of cancer cells and metastatic progression. The study aims to investigate the roles of TAZ and YAP in anoikis resistance in basal-like (BL) TNBC cells, which comprise a major subtype (>70%) of TNBC. We found that TAZ and YAP had nonredundant roles in anchorage-independent cancer cell survival or anoikis resistance. Particularly, TAZ was indispensable for anoikis resistance in BL-TNBC cells but not for survival of non-transformed mammary epithelial cells (MECs). In contrast, YAP, a paralog of TAZ, was indispensable for survival of both non-transformed MECs and cancer cells. Therefore, TAZ might be a preferable therapeutic target against dissemination of aggressive cancer cells without killing normal cells. Interestingly, TAZ was abnormally stabilized in BL-TNBC cells under non-adherent conditions, which promoted anoikis resistance. Furthermore, OTUB1, a deubiquitinating enzyme, was responsible for the stabilization of TAZ in detached BL-TNBC cells. Importantly, simultaneous high expression of TAZ and OTUB1 was associated with poor prognosis in BC. Thus, OTUB1 has emerged as a potentially druggable target. Successful inhibition of OTUB1 enzymatic activity is expected to downregulate TAZ and eventually prevents metastasis of aggressive cancers, such as BL-TNBC.

Pathogen resistance in soils associated with bacteriome network reconstruction through reductive soil disinfestation.

Reductive soil disinfestation (RSD) is an effective bioremediation technique to restructure the soil microbial community and eliminate soilborne phytopathogens. Yet we still lack a comprehensive understanding of the keystone taxa involved and their roles in ecosystem functioning in degraded soils treated by RSD. In this study, the bacteriome network structure in RSD-treated soil and the subsequent cultivation process were explored. As a result, bacterial communities in RSD-treated soil developed more complex topologies and stable co-occurrence patterns. The richness and diversity of keystone taxa were higher in the RSD group (module hub: 0.57%; connector: 23.98%) than in the Control group (module hub: 0.16%; connector: 19.34%). The restoration of keystone taxa in RSD-treated soil was significantly (P < 0.01) correlated with soil pH, total organic carbon, and total nitrogen. Moreover, a strong negative correlation (r = -0.712; P < 0.01) was found between keystone taxa richness and Fusarium abundance. Our results suggest that keystone taxa involved in the RSD network structure are capable of maintaining a flexible generalist mode of metabolism, namely with respect to nitrogen fixation, methylotrophy, and methanotrophy. Furthermore, distinct network modules composed by numerous anti-pathogen agents were formed in RSD-treated soil; i.e., the genera Hydrogenispora, Azotobacter, Sphingomonas, and Clostridium_8 under the soil treatment stage, and the genera Anaerolinea and Pseudarthrobacter under the plant cultivation stage. The study provides novel insights into the association between fungistasis and keystone or sensitive taxa in RSD-treated soil, with significant implications for comprehending the mechanisms of RSD. KEY POINTS: • RSD enhanced bacteriome network stability and restored keystone taxa. • Keystone taxa richness was negatively correlated with Fusarium abundance. • Distinct sensitive OTUs and modules were formed in RSD soil.

Plant deubiquitinases: from structure and activity to biological functions.

KEY MESSAGE: This article attempts to provide comprehensive review of plant deubiquitinases, paying special attention to recent advances in their biochemical activities and biological functions. Proteins in eukaryotes are subjected to post-translational modifications, in which ubiquitination is regarded as a reversible process. Cellular deubiquitinases (DUBs) are a key component of the ubiquitin (Ub)-proteasome system responsible for cellular protein homeostasis. DUBs recycle Ub by hydrolyzing poly-Ub chains on target proteins, and maintain a balance of the cellular Ub pool. In addition, some DUBs prefer to cleave poly-Ub chains not linked through the conventional K48 residue, which often alter the substrate activity instead of its stability. In plants, all seven known DUB subfamilies have been identified, namely Ub-binding protease/Ub-specific protease (UBP/USP), Ub C-terminal hydrolase (UCH), Machado-Joseph domain-containing protease (MJD), ovarian-tumor domain-containing protease (OTU), zinc finger with UFM1-specific peptidase domain protease (ZUFSP), motif interacting with Ub-containing novel DUB family (MINDY), and JAB1/MPN/MOV34 protease (JAMM). This review focuses on recent advances in the structure, activity, and biological functions of plant DUBs, particularly in the model plant Arabidopsis.

The Arabidopsis Deubiquitylase OTU5 Suppresses Flowering by Histone Modification-Mediated Activation of the Major Flowering Repressors FLC, MAF4, and MAF5.

Distinct phylogeny and substrate specificities suggest that 12 Arabidopsis Ovarian Tumor domain-containing (OTU) deubiquitinases participate in conserved or plant-specific functions. The otu5-1 null mutant displayed a pleiotropic phenotype, including early flowering, mimicking that of mutants harboring defects in subunits (e.g., ARP6) of the SWR1 complex (SWR1c) involved in histone H2A.Z deposition. Transcriptome and RT-qPCR analyses suggest that downregulated FLC and MAF4-5 are responsible for the early flowering of otu5-1. qChIP analyses revealed a reduction and increase in activating and repressive histone marks, respectively, on FLC and MAF4-5 in otu5-1. Subcellular fractionation, GFP-fusion expression, and MNase treatment of chromatin showed that OTU5 is nucleus-enriched and chromatin-associated. Moreover, OTU5 was found to be associated with FLC and MAF4-5. The OTU5-associated protein complex(es) appears to be distinct from SWR1c, as the molecular weights of OTU5 complex(es) were unaltered in arp6-1 plants. Furthermore, the otu5-1 arp6-1 double mutant exhibited synergistic phenotypes, and H2A.Z levels on FLC/MAF4-5 were reduced in arp6-1 but not otu5-1. Our results support the proposition that Arabidopsis OTU5, acting independently of SWR1c, suppresses flowering by activating FLC and MAF4-5 through histone modification. Double-mutant analyses also indicate that OTU5 acts independently of the HUB1-mediated pathway, but it is partially required for FLC-mediated flowering suppression in autonomous pathway mutants and FRIGIDA-Col.

Structural Basis of Ubiquitin Recognition by a Bacterial Ovarian Tumor Deubiquitinase LotA.

Pathogenic bacteria have acquired a vast array of eukaryotic-protein-like proteins via intimate interaction with host cells. Bacterial effector proteins that function as ubiquitin ligases and deubiquitinases (DUBs) are remarkable examples of such molecular mimicry. LotA, a Legionella pneumophila effector, belongs to the ovarian tumor (OTU) superfamily, which regulates diverse ubiquitin signals by their DUB activities. LotA harbors two OTU domains that have distinct reactivities; the first one is responsible for the cleavage of the K6-linked ubiquitin chain, and the second one shows an uncommon preference for long chains of ubiquitin. Here, we report the crystal structure of a middle domain of LotA (LotAM), which contains the second OTU domain. LotAM consists of two distinct subdomains, a catalytic domain having high structural similarity with human OTU DUBs and an extended helical lobe (EHL) domain, which is characteristically conserved only in Legionella OTU DUBs. The docking simulation of LotAM with ubiquitin suggested that hydrophobic and electrostatic interactions between the EHL of LotAM and the C-terminal region of ubiquitin are crucial for the binding of ubiquitin to LotAM. The structure-based mutagenesis demonstrated that the acidic residue in the characteristic short helical segment termed the "helical arm" is essential for the enzymatic activity of LotAM. The EHL domain of the three Legionella OTU DUBs, LotA, LotB, and LotC, share the "helical arm" structure, suggesting that the EHL domain defines the Lot-OTUs as a unique class of DUBs. IMPORTANCE To successfully colonize, some pathogenic bacteria hijack the host ubiquitin system. Legionella OTU-like-DUBs (Lot-DUBs) are novel bacterial deubiquitinases found in effector proteins of L. pneumophila. LotA is a member of Lot-DUBs and has two OTU domains (OTU1 and OTU2). We determined the structure of a middle fragment of LotA (LotAM), which includes OTU2. LotAM consists of the conserved catalytic domain and the Legionella OTUs-specific EHL domain. The docking simulation with ubiquitin and the mutational analysis suggested that the acidic surface in the EHL is essential for enzymatic activity. The structure of the EHL differs from those of other Lot-DUBs, suggesting that the variation of the EHL is related to the variable cleaving specificity of each DUB.

Substrate-assisted activation and selectivity of the bacterial RavD effector deubiquitinylase.

Deubiquitinylases (DUBs) catalyze the peptide bond cleavage of specific ubiquitin linkages at distinct protein substrates. Pathogens from viruses and bacteria independently developed effector proteins with DUB activity to mimic host DUB functions and circumvent immune responses. The effector protein RavD from Legionella pneumophila cleaves linear ubiquitin chains with an exclusive methionine-1 selectivity. It thus performs as a functional analogue of the human DUB OTULIN, which achieves its selectivity only via a specialized proximal ubiquitin S1' binding site as well as a substrate-assisted activation of the catalytic triad. An analysis of the crystal structures of bacterial RavD in its free and di-ubiquitin-bound forms, in order to rationalize the structural basis for its selectivity and activation mechanism, is not fully conclusive. As these ambiguities might arise from the introduced double mutation of the di-ubiquitin substrate in the RavD-di-ubiquitin complex crystal structure, biomolecular modeling, and molecular dynamics sampling (1-2 µs for each system of RavD and OTULIN) were employed to reconstitute the physiological RavD-di-ubiquitin complex. The simulations show that the distal S1 ubiquitin binding sites of RavD and OTULIN are similar in terms of interface area, composition, and ubiquitin binding affinity. The proximal S1' site of RavD, in contrast, is significantly smaller and ubiquitin binding is weaker and more flexible than in OTULIN. Upon substrate access, the residues of the catalytic triad of RavD show a reduction of flexibility and a conformational transition toward a catalytically active state. Thus, the enzymatic activation of RavD is presumably also substrate-assisted and a clear rationale for the common M1-substrate selectivity.

Discovery of a small-molecule inhibitor targeting the ovarian tumor domain of a novel Tamdy orthonairoviruse associated with human febrile illness.

Tick-borne orthonairoviruses have been characterized as a global health threat to humans and animals. Tacheng Tick virus 1 (TcTV-1) from this family was provided as evidence that is associated with the febrile illness syndrome. Here, we first identify and demonstrate that the ovarian tumor (OTU) domain of TcTV-1 has remarkable deubiquitinating activity both in vitro and in vivo. By solving the crystal structure of TcTV-1 OTU (tcOTU) domain and comparing it to that of human deubiquitinating enzymes, we found that overall structures of tcOTU and human OTU family are similar, but the residues involved in the catalytic pocket vary widely. Based on the tcOTU domain we screened 5090 bioactive compounds and found mecobalamin had a good effect on suppressing the deubiquitinating activity. The structural model of tcOTU and mecobalamin suggests that mecobalamin occupies the site of the substrate Ub, by blocking the substrate binding to the enzyme. Thus, our results showed OTU domain of TcTV-1 has a robust deubiquitinating activity and mecobalamin or its derivatives might be promising candidates for the treatment or prevention of disease caused by the TcTV-1 virus.

Hawaiian Fungal Amplicon Sequence Variants Reveal Otherwise Hidden Biogeography.

To study biogeography and other ecological patterns of microorganisms, including fungi, scientists have been using operational taxonomic units (OTUs) as representations of species or species hypotheses. However, when defined by 97% sequence similarity cutoff at an accepted barcode locus such as 16S in bacteria or ITS in fungi, these OTUs can obscure biogeographic patterns, mask taxonomic diversity, and hinder meta-analyses. Amplicon sequence variants (ASVs) have been proposed to alleviate all of these issues and have been shown to do so in bacteria. Analyzing ASVs is just emerging as a common practice among fungal studies, and it is unclear whether the benefits found in bacterial studies of using such an approach carryover to fungi. Here, we conducted a meta-analysis of Hawaiian fungi by analyzing ITS1 amplicon sequencing data as ASVs and exploring ecological patterns. These surveys spanned three island groups and five ecosystems combined into the first comprehensive Hawaiian Mycobiome ASV Database. Our results show that ASVs can be used to combine fungal ITS surveys, increase reproducibility, and maintain the broad ecological patterns observed with OTUs, including diversity orderings. Additionally, the ASVs that comprise some of the most common OTUs in our database reveals some island specialists, indicating that traditional OTU clustering can obscure important biogeographic patterns. We recommend that future fungal studies, especially those aimed at assessing biogeography, analyze ASVs rather than OTUs. We conclude that similar to bacterial studies, ASVs improve reproducibility and data sharing for fungal studies.

Effects of dietary fibre on intestinal microbiota in geese evaluated by 16SrRNA gene sequencing.

AIMS: The purpose of the research is to study the effects of different fibre types and sources on the intestinal flora of geese. METHODS AND RESULTS: A total of 48 geese (males: 35 days old) were divided into four groups, each of which included three replicates of four geese. Groups 1-4 were fed a diet containing 5% corn stover Crude fibre (CF, the LJ group), 8% corn stover CF (the HJ group), 5% alfalfa CF (the LM group) or 8% alfalfa CF (the HM group), respectively. After 42 days of feeding, the intestinal flora of each group was determined by 16SrRNA gene sequencing. In the duodenum, the diet supplemented with corn stover meal increased the relative abundance of Proteobacteria, Actinobacteria and Euryarchaeota, and with alfalfa as fibre source increased the relative abundance of Firmicutes, Bacteroidetes, Tenericutes and Chloroflexi. In the jejunum, Bacteroidetes, Actinobacteria, Planctomycetes, Acidobacteria, Tenericutes and Spirochetes were significantly more abundant in the corn stover group. There were no significant differences among the results for the other two fibre sources, which were fibre level in their influence where in ileum. Firmicutes, Deferribacteres and Euryarchaeota with corn stover as fibre source in the cecum were higher than the alfalfa group. CONCLUSIONS: Different fibre sources have significant effects on goose gut microbiota. The same flora has the same trend of change in different intestinal segments. The relative fibre source in the ileum makes the gut microbiota more sensitive to differences in fibre levels. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proved that the dietary fibre affects the intestinal flora. At the same time, different groups of dietary fibre may be used to provide the possibility to study functional roles of specific bacteria in host physiology.

Response of human gut microbiota under simulated microgravity.

The present study was conducted to investigate the influence of microgravity on human gut microbiota using 16S rRNA gene sequencing in vitro. The diamagnetic material magnetic levitation method was used to simulate weightless environment. The human gut microbiota was cultured under two different conditions: normal gravity (1 g), and simulated microgravity (0 g), which showed that both the richness (P = 0.04) and diversity (P = 0.0002) of human gut microbiota were significantly altered. As compared to the normal gravity, the simulated microgravity significantly reduced abundance of bacteria related to anti-inflammatory effects, such as Subdoligranulum, Faecalibacterium, Fusicatenibacter, Butyricicoccus, and Lachnospiraceae-NK4A136-0 group (P < 0.05), while significantly increased that of Alistipes and Eubacterium-Ventriosum-group (P < 0.05). Moreover, the Spearman's correlation analysis showed that there were more significantly correlated species (|r|≥ 0.5, P < 0.05) in normal gravity than that in the simulated microgravity. KEGG pathway analysis revealed that the microgravity significantly (P < 0.05) affected the metabolism of gut microbiota, such as the metabolism of pyrimidine, fatty acids, glyoxylate and dicarboxylate, peptidoglycan biosynthesis, and carbon fixation in photosynthetic organisms. These results suggested that the exposure to a microgravity environment might induce disturbances in human gut microbiota. KEY POINTS: • Using 16sRNA gene sequencing technology, it was found that magnetic levitation-simulated microgravity had varying degrees of influence on the abundance, diversity, species correlation, and KEGG pathways of human intestinal microbes. • Digital PCR can improve the detection rate of microorganisms with low abundance.

Identification of first-in-class plasmodium OTU inhibitors with potent anti-malarial activity.

OTU proteases antagonize the cellular defense in the host cells and involve in pathogenesis. Intriguingly, P. falciparum, P. vivax, and P. yoelii have an uncharacterized and highly conserved viral OTU-like proteins. However, their structure, function or inhibitors have not been previously reported. To this end, we have performed structural modeling, small molecule screening, deconjugation assays to characterize and develop first-in-class inhibitors of P. falciparum, P. vivax, and P. yoelii OTU-like proteins. These Plasmodium OTU-like proteins have highly conserved residues in the catalytic and inhibition pockets similar to viral OTU proteins. Plasmodium OTU proteins demonstrated Ubiquitin and ISG15 deconjugation activities as evident by intracellular ubiquitinated protein content analyzed by western blot and flow cytometry. We screened a library of small molecules to determine plasmodium OTU inhibitors with potent anti-malarial activity. Enrichment and correlation studies identified structurally similar molecules. We have identified two small molecules that inhibit P. falciparum, P. vivax, and P. yoelii OTU proteins (IC50 values as low as 30 nM) with potent anti-malarial activity (IC50 of 4.1-6.5 µM). We also established enzyme kinetics, druglikeness, ADME, and QSAR model. MD simulations allowed us to resolve how inhibitors interacted with plasmodium OTU proteins. These findings suggest that targeting malarial OTU-like proteases is a plausible strategy to develop new anti-malarial therapies.

Activation and selectivity of OTUB-1 and OTUB-2 deubiquitinylases.

The ovarian tumor domain (OTU) deubiquitinylating cysteine proteases OTUB1 and OTUB2 (OTU ubiquitin aldehyde binding 1 and 2) are representative members of the OTU subfamily of deubiquitinylases. Deubiquitinylation critically regulates a multitude of important cellular processes, such as apoptosis, cell signaling, and growth. Moreover, elevated OTUB expression has been observed in various cancers, including glioma, endometrial cancer, ovarian cancer, and breast cancer. Here, using molecular dynamics simulation approaches, we found that both OTUB1 and OTUB2 display a catalytic triad characteristic of proteases but differ in their configuration and protonation states. The OTUB1 protein had a prearranged catalytic site, with strong electrostatic interactions between the active-site residues His265 and Asp267 In OTUB2, however, the arrangement of the catalytic triad was different. In the absence of ubiquitin, the neutral states of the catalytic-site residues in OTUB2 were more stable, resulting in larger distances between these residues. Only upon ubiquitin binding did the catalytic triad in OTUB2 rearrange and bring the active site into a catalytically feasible state. An analysis of water access channels revealed only a few diffusion trajectories for the catalytically active form of OTUB1, whereas in OTUB2 the catalytic site was solvent-accessible, and a larger number of water molecules reached and left the binding pocket. Interestingly, in OTUB2, the catalytic residues His224 and Asn226 formed a stable hydrogen bond. We propose that the observed differences in activation kinetics, protonation states, water channels, and active-site accessibility between OTUB1 and OTUB2 may be relevant for the selective design of OTU inhibitors.

Amyloid aggregates of the deubiquitinase OTUB1 are neurotoxic, suggesting that they contribute to the development of Parkinson's disease.

Parkinson's disease (PD) is a multifactorial malady and the second most common neurodegenerative disorder, characterized by loss of dopaminergic neurons in the midbrain. A hallmark of PD pathology is the formation of intracellular protein inclusions, termed Lewy bodies (LBs). Recent MS studies have shown that OTU deubiquitinase ubiquitin aldehyde-binding 1 (OTUB1), a deubiquitinating enzyme of the OTU family, is enriched together with α-synuclein in LBs from individuals with PD and is also present in amyloid plaques associated with Alzheimer's disease. In the present study, using mammalian cell cultures and a PD mouse model, along with CD spectroscopy, atomic force microscopy, immunofluorescence-based imaging, and various biochemical assays, we demonstrate that after heat-induced protein aggregation, OTUB1 reacts strongly with both anti-A11 and anti-osteocalcin antibodies, detecting oligomeric, prefibrillar structures or fibrillar species of amyloidogenic proteins, respectively. Further, recombinant OTUB1 exhibited high thioflavin-T and Congo red binding and increased ß-sheet formation upon heat induction. The oligomeric OTUB1 aggregates were highly cytotoxic, characteristic of many amyloid proteins. OTUB1 formed inclusions in neuronal cells and co-localized with thioflavin S and with α-synuclein during rotenone-induced stress. It also co-localized with the disease-associated variant pS129-α-synuclein in rotenone-exposed mouse brains. Interestingly, OTUB1 aggregates were also associated with severe cytoskeleton damage, rapid internalization inside the neuronal cells, and mitochondrial damage, all of which contribute to neurotoxicity. In conclusion, the results of our study indicate that OTUB1 may contribute to LB pathology through its amyloidogenic properties.

Oxygen-dependent asparagine hydroxylation of the ubiquitin-associated (UBA) domain in Cezanne regulates ubiquitin binding.

Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain-containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn35 within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBACez), which lacks conserved UBA domain properties. We show that UBACez binds Lys11-, Lys48-, Lys63-, and Met1-linked ubiquitin chains in vitro, establishing UBACez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBACez with ubiquitin is mediated via a noncanonical surface and that hydroxylation of Asn35 inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBACez Our results indicate that UBACez can indeed fulfill this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.

Alterations in Intestinal Microbiota of Children With Celiac Disease at the Time of Diagnosis and on a Gluten-free Diet.

BACKGROUND AND AIMS: It is not clear whether alterations in the intestinal microbiota of children with celiac disease (CD) cause the disease or are a result of disease and/or its treatment with a gluten-free diet (GFD). METHODS: We obtained 167 fecal samples from 141 children (20 with new-onset CD, 45 treated with a GFD, 57 healthy children, and 19 unaffected siblings of children with CD) in Glasgow, Scotland. Samples were analyzed by 16S ribosomal RNA sequencing, and diet-related metabolites were measured by gas chromatography. We obtained fecal samples from 13 children with new-onset CD after 6 and 12 months on a GFD. Relationships between microbiota with diet composition, gastrointestinal function, and biomarkers of GFD compliance were explored. RESULTS: Microbiota α diversity did not differ among groups. Microbial dysbiosis was not observed in children with new-onset CD. In contrast, 2.8% (Bray-Curtis dissimilarity index, P = .025) and 2.5% (UniFrac distances, P = .027) of the variation in microbiota composition could be explained by the GFD. Between 3% and 5% of all taxa differed among all group comparisons. Eleven distinctive operational taxonomic units composed a microbe signature specific to CD with high diagnostic probability. Most operational taxonomic units that differed between patients on a GFD with new-onset CD vs healthy children were associated with nutrient and food group intake (from 75% to 94%) and with biomarkers of gluten ingestion. Fecal levels of butyrate and ammonia decreased during the GFD. CONCLUSIONS: Although several alterations in the intestinal microbiota of children with established CD appear to be effects of a GFD, specific bacteria were found to be distinct biomarkers of CD. Studies are needed to determine whether these bacteria contribute to pathogenesis of CD.