Activity-Guided Proteomic Profiling of Proteasomes Uncovers a Variety of Active (and Inactive) Proteasome Species.

Proteasomes are multisubunit, multicatalytic protein complexes present in eukaryotic cells that degrade misfolded, damaged, or unstructured proteins. In this study, we used an activity-guided proteomic methodology based on a fluorogenic peptide substrate to characterize the composition of proteasome complexes in WT yeast and the changes these complexes undergo upon the deletion of Pre9 (Δα3) or of Sem1 (ΔSem1). A comparison of whole-cell proteomic analysis to activity-guided proteasome profiling indicates that the amounts of proteasomal proteins and proteasome interacting proteins in the assembled active proteasomes differ significantly from their total amounts in the cell as a whole. Using this activity-guided profiling approach, we characterized the changes in the abundance of subunits of various active proteasome species in different strains, quantified the relative abundance of active proteasomes across these strains, and charted the overall distribution of different proteasome species within each strain. The distributions obtained by our mass spectrometry-based quantification were markedly higher for some proteasome species than those obtained by activity-based quantification alone, suggesting that the activity of some of these species is impaired. The impaired activity appeared mostly among 20SBlm10 proteasome species which account for 20% of the active proteasomes in WT. To identify the factors behind this impaired activity, we mapped and quantified known proteasome-interacting proteins. Our results suggested that some of the reduced activity might be due to the association of the proteasome inhibitor Fub1. Additionally, we provide novel evidence for the presence of nonmature and therefore inactive proteasomal protease subunits ß2 and ß5 in the fully assembled proteasomes.

Rational design of proteasome inhibitors based on the structure of the endogenous inhibitor PI31/Fub1.

Proteasome inhibitors are widely used anticancer drugs. The three clinically approved agents are modified small peptides that preferentially target one of the proteasome's three active sites (ß5) at physiologic concentrations. In addition to these drugs, there is also an endogenous proteasome inhibitor, PI31/Fub1, that enters the proteasome's interior to simultaneously yet specifically inhibit all three active sites. Here, we have used PI31's evolutionarily optimized inhibitory mechanisms to develop a suite of potent and specific ß2 inhibitors. The lead compound strongly inhibited growth of multiple myeloma cells as a standalone agent, indicating the compound's cell permeability and establishing ß2 as a potential therapeutic target in multiple myeloma. The lead compound also showed strong synergy with the existing ß5 inhibitor bortezomib; such combination therapies might help with existing challenges of resistance and severe side effects. These results represent an effective method for rational structure-guided development of proteasome inhibitors.

Transcriptional Readthrough Interrupts Boundary Function in Drosophila.

In higher eukaryotes, distance enhancer-promoter interactions are organized by topologically associated domains, tethering elements, and chromatin insulators/boundaries. While insulators/boundaries play a central role in chromosome organization, the mechanisms regulating their functions are largely unknown. In the studies reported here, we have taken advantage of the well-characterized Drosophila bithorax complex (BX-C) to study one potential mechanism for controlling boundary function. The regulatory domains of BX-C are flanked by boundaries, which block crosstalk with their neighboring domains and also support long-distance interactions between the regulatory domains and their target gene. As many lncRNAs have been found in BX-C, we asked whether readthrough transcription (RT) can impact boundary function. For this purpose, we took advantage of two BX-C boundary replacement platforms, Fab-7attP50 and F2attP, in which the Fab-7 and Fub boundaries, respectively, are deleted and replaced with an attP site. We introduced boundary elements, promoters, and polyadenylation signals arranged in different combinations and then assayed for boundary function. Our results show that RT can interfere with boundary activity. Since lncRNAs represent a significant fraction of Pol II transcripts in multicellular eukaryotes, it is therefore possible that RT may be a widely used mechanism to alter boundary function and regulation of gene expression.

Sex-dependent pharmacological profiles of the synthetic cannabinoid MMB-Fubinaca.

Synthetic cannabinoids have emerged as novel psychoactive substances with damaging consequences for public health. They exhibit high affinity at the cannabinoid type-1 (CB1 ) receptor and produce similar and often more potent effects as other CB1 receptor agonists. However, we are still far from a complete pharmacological understanding of these compounds. In this study, by using behavioral, molecular, pharmacological, and electrophysiological approaches, we aimed at characterizing several in vitro and in vivo pharmacological effects of the synthetic cannabinoid MMB-Fubinaca (also known as AMB-Fubinaca or FUB-AMB), a particular synthetic cannabinoid. MMB-Fubinaca stimulates CB1 receptor-mediated functional coupling to G-proteins in mouse and human brain preparations in a similar manner as the CB1 receptor agonist WIN55,512-2 but with a much greater potency. Both drugs similarly activate the CB1 receptor-dependent extracellular signal-regulated kinase (ERK) pathway. Notably, in vivo administration of MMB-Fubinaca in mice induced greater behavioral and electrophysiological effects in male than in female mice in a CB1 receptor-dependent manner. Overall, these data provide a solid pharmacological profiling of the effects of MMB-Fubinaca and important information about the mechanisms of action underlying its harmful impact in humans. At the same time, they reinforce the significant sexual dimorphism of cannabinoid actions, which will have to be taken into account in future animal and clinical studies.

Assessment of synthetic cannabinoid FUB-AMB and its ester hydrolysis metabolite in human liver microsomes and human blood samples using UHPLC-MS/MS.

FUB-AMB, an indazole carboxamide synthetic cannabinoid recreational drug, was one of the compounds most frequently reported to governmental agencies worldwide between 2016 and 2019. It has been implicated in intoxications and fatalities, posing a risk to public health. In the current study, FUB-AMB was incubated with human liver microsomes (HLM) to assess its metabolic fate and stability and to determine if its major ester hydrolysis metabolite (M1) was present in 12 authentic forensic human blood samples from driving under the influence of drug cases and postmortem investigations using UHPLC-MS/MS. FUB-AMB was rapidly metabolized in HLM, generating M1 that was stable through a 120-min incubation period, a finding that indicates a potential long detection window in human biological samples. M1 was identified in all blood samples, and no parent drug was detected. The authors propose that M1 is a reliable marker for inclusion in laboratory blood screens for FUB-AMB; this metabolite may be pharmacologically active like its precursor FUB-AMB. M1 frequently appears in samples in which the parent drug is undetectable and can point to the causative agent. The results suggest that it is imperative that synthetic cannabinoid laboratory assay panels include metabolites, especially known or potential pharmacologically active metabolites, particularly for compounds with short half-lives.

Uveitis Registries - A Digital Tool for Patient Care, Education, Research, and Collaboration.

PURPOSE: Clinical registries are increasingly important in research and clinical advancement. This review explores and compares current uveitis registries and recommends future directions on how uveitis registries can complement one another for synergistic effect and benefit. METHODS: From a systematic search, 861 citations were screened for longitudinal, non-interventional, and multicenter uveitis-specific registries. Additional registries were identified via consultations with uveitis experts. Characteristics of all registries were analyzed and compared. RESULTS: Four registries were identified: Treatment Exit Options for Non-infectious Uveitis, AutoInflammatory Disease Alliance International Registry, Ocular Autoimmune Systemic Inflammatory Infectious Study, and Fight Uveitis Blindness!. Despite certain differences, these registries have the overarching goal of collecting large quantities of real-world, high-quality patient data to improve the understanding of uveitis. CONCLUSION: The four uveitis registries share similar goals and collect clinical data from overlapping geographical regions. There is vast potential for collaboration, including data sharing to further augment datasets for analysis.

lncRNA read-through regulates the BX-C insulator Fub-1.

Though long non-coding RNAs (lncRNAs) represent a substantial fraction of the Pol II transcripts in multicellular animals, only a few have known functions. Here we report that the blocking activity of the Bithorax complex (BX-C) Fub-1 boundary is segmentally regulated by its own lncRNA. The Fub-1 boundary is located between the Ultrabithorax (Ubx) gene and the bxd/pbx regulatory domain, which is responsible for regulating Ubx expression in parasegment PS6/segment A1. Fub-1 consists of two hypersensitive sites, HS1 and HS2. HS1 is an insulator while HS2 functions primarily as an lncRNA promoter. To activate Ubx expression in PS6/A1, enhancers in the bxd/pbx domain must be able to bypass Fub-1 blocking activity. We show that the expression of the Fub-1 lncRNAs in PS6/A1 from the HS2 promoter inactivates Fub-1 insulating activity. Inactivation is due to read-through as the HS2 promoter must be directed toward HS1 to disrupt blocking.

Blocking the blocker.

An unexpected interaction between a long non-coding RNA locus and a genetic insulator called Fub-1 has an important role in gene regulation during development in Drosophila.

Deciphering the effect of FUB1 disruption on fusaric acid production and pathogenicity in Fusarium circinatum.

Fusarium circinatum is an important pathogen of pine trees. However, little is known regarding the molecular processes underlying its pathogenesis. We explored the potential role of the phytotoxin fusaric acid (FA) in the pathogenicity of the fungus. FA is produced by products of the FUB biosynthesis gene cluster, containing FUB1-12. Of these, FUB1 encodes the core polyketide synthase, which we disrupted. We used the resulting mutant strain to investigate whether FUB1 and FA production play a role in the virulence of F. circinatum on pine. Our results showed that FA production was abolished both in vitro and in planta. However, bikaverin production was increased in the knockout mutant. FUB1 disruption also corresponded with downregulation of a F. circinatum homologue of LaeA, a master transcriptional regulator of secondary metabolism. Lesion lengths produced by the FUB1 knockout mutant on inoculated Pinus patula seedlings were significantly smaller than those produced by the wild type strain. Collectively, these results show that FUB1 plays a role in FA production in F. circinatum, and that this gene contributes to the aggressiveness of F. circinatum on P. patula. This study will contribute to the limited knowledge we have about the molecular basis of pathogenicity in this fungus.

Establishment of a brain cell line (FuB-1) from mummichog (Fundulus heteroclitus) and its application to fish virology, immunity and nanoplastics toxicology.

The marine fish mummichog (Fundulus heteroclitus), extensively used as research model, including in ecotoxicology, for over a century has been surpassed by other fish species. This fact may be associated with the lack of cell lines from this species, excellent models for the comprehension of fish physiology, immunology, toxicology and virology, that contribute to the reduction in the number of animals used in research. We have generated, for the first time, a brain-derived cell line from mummichog, FuB-1, and evaluated its application to the fields of fish virology, immunity and toxicology. First, FuB-1 cells show epithelial morphology and neural stem/astroglial origin. Secondly, FuB-1 cells effectively supports the replication of both spring viremia carp (SVCV) and infectious pancreatic necrosis (IPNV) viruses, but not nodavirus (NNV), indicating its potential use for fish virology. Related to this, FuB-1 cells infected with NNV up-regulate the transcription of genes related to the antiviral immune response, leading to cell resistance; while they are unaltered when infected with IPNV and SVCV, facilitating viral replication. Finally, FuB-1 cells were used for toxicological purposes and we demonstrated that exposure to either polystyrene nanoplastics (PS-100) or several human-usage pharmaceuticals are cytotoxic. Additionally, PS-100 particles increase the antioxidant catalase and glutathione S-transferase activities and decrease the total non-protein thiols in FuB-1 cells. However, PS-100 particles are able to reduce the cytotoxic effects induced by the pharmaceuticals. In conclusion, we have generated a cell line from mummichog, which might represent a valuable model for fish studies in the fields of virology, immunology and toxicology.

Extraordinary long detection window of a synthetic cannabinoid metabolite in human urine - Potential impact on therapeutic decisions.

Synthetic cannabinoids (SCs) have become established drugs of abuse. They play an increasing role in drug therapy, where abstinence control testing is required. Differentiation between recent drug uptake and uptake in the distant past is important for drug therapy. This study aimed to evaluate the detection window of a metabolite commonly used as a consumption marker for AB-FUBINACA and AMB-FUBINACA (synonym: FUB-AMB) in urine analysis. The acidic hydrolysis metabolite was quantified in urine samples of a drug user by applying a validated analytical method. The concentration profile of the metabolite was correlated with usage data of the subject. Pharmacokinetic properties of AB-FUBINACA were collected by analysis of serum and urine samples from a controlled administration study (single oral ingestion of AB-FUBINACA). Thirteen urine samples were taken without advance notice over 2 years. The metabolite was detected in the first urine sample at 0.77 ng/mg creatinine and subsequently in concentrations ranging from 0.06 to 0.29 ng/mg creatinine. Usage data showed credible abstinence from SCs during this period. The pharmacokinetic properties observed within the controlled self-administration study supported the hypothesis of distribution into deeper compartments and long-lasting elimination (serum concentration-time curve showing biphasic kinetics). An elimination phase of over 1 year after the last drug uptake seems plausible in cases of extensive consumption. To avoid misinterpretation of positive findings, we recommend testing patients with known SC use at the beginning of the abstinence program and to re-test continuously at short time intervals. These data enable the correct interpretation of analytical findings.

A case of 5F-ADB / FUB-AMB abuse: Drug-induced or drug-related death?

Synthetic cannabinoids (SCs) belong to the group of new psychoactive substances (NPS) which appear sprayed on herbal mixtures on the "street" drug market and are intended for smoking like marijuana. In the present report we discuss a fatal case of 18-years-old boy, who had smoked SCs since several months and an overuse of SCs during last 48 h of his life has been apprised. The autopsy findings revealed acute respiratory distress syndrome (ARDS). Both toxicological analysis of deceased blood and urine samples and chemical analysis of the herbal mixture seized revealed presence of two SCs - 5F-ADB and FUB-AMB. The amount of 5F-ADB in blood was found to be 3.7 ng/mL by standard addition method. Severe and irreversible morphology changes in lung specimen, leading to ischemic damage of all internal organs and tissues, were observed during histological examination. The present case can be discussed as an example of both drug-induced and drug-related death resulting from acute intoxication with 5F-ADB and FUB-AMB as well as from systematic use of both synthetic cannabinoids.

Identification of synthetic cannabinoids that were seized, consumed, or associated with deaths in Kuwait in 2018 using GC-MS and LC-MS-MS analysis.

Synthetic cannabinoids are gaining much popularity worldwide. Although the death rate associated with their use is rising, these drugs are the largest and fastest growing class of novel psychoactive substances. Despite increased concerns regarding adverse effects stemming from the use of synthetic cannabinoids, there is no published data on the subject for the Gulf region or Kuwait, specifically. The current study investigates the diversity of synthetic cannabinoids in Kuwait in 2018. In total, 434 cases from the Narcotics and Psychotropic Laboratory, 70 cases from the Toxicology Laboratory, and six cases from the Forensic Medicine Department were reviewed and analyzed. Numerous synthetic cannabinoid types were identified using GC-MS and LC-MS-MS. The majority of synthetic cannabinoids were members of the indazole-3-carboxamide or indole-3-carboxamide families. Members from the indazole-3-carboxamide family identified in Kuwait were 5F-ADB, FUB-AMB, ADB-FUBINACA, AB-FUBINACA, 5F-ADB-PINACA, 5F-AKB-48, 5Cl-AKB-48, MDMB-FUBINACA, 5F-AB-PINACA, APINACA, and AB-PINACA whereas MDMB-CHMICA, 5F-MDMB-PICA, ADB-BICA, and MMB-CHMICA belonged to the indole-3-carboxamide family. In addition, members of other families were identified, including CBL2201 and UR-144, which belonged to indole-3-carboxylate and cyclopropylindole families, respectively. The most common synthetic cannabinoids were 5F-ADB, FUB-AMB, and 5Cl-AKB-48. Various mixes of two, three, or four types of synthetic cannabinoids were identified, and mixtures of synthetic cannabinoids with other illicit drugs were also present. Our findings show that in Kuwait, the most common mix of synthetic cannabinoids is FUB-AMB with 5F-ADB. These two types were mixed, either together or individually, with methamphetamine, tramadol, heroin, Δ9THC, and ketamine. Most importantly, our results reveal the synthetic cannabinoid types that were associated with six reported deaths.

In Vitro and In Vivo Human Metabolism of Synthetic Cannabinoids FDU-PB-22 and FUB-PB-22.

In 2014, FDU-PB-22 and FUB-PB-22, two novel synthetic cannabinoids, were detected in herbal blends in Japan, Russia, and Germany and were quickly added to their scheduled drugs list. Unfortunately, no human metabolism data are currently available, making it challenging to confirm their intake. The present study aims to identify appropriate analytical markers by investigating FDU-PB-22 and FUB-PB-22 metabolism in human hepatocytes and confirm the results in authentic urine specimens. For metabolic stability, 1 µM FDU-PB-22 and FUB-PB-22 was incubated with human liver microsomes for up to 1 h; for metabolite profiling, 10 µM was incubated with human hepatocytes for 3 h. Two authentic urine specimens from FDU-PB-22 and FUB-PB-22 positive cases were analyzed after ß-glucuronidase hydrolysis. Metabolite identification in hepatocyte samples and urine specimens was accomplished by high-resolution mass spectrometry using information-dependent acquisition. Both FDU-PB-22 and FUB-PB-22 were rapidly metabolized in HLM with half-lives of 12.4 and 11.5 min, respectively. In human hepatocyte samples, we identified seven metabolites for both compounds, generated by ester hydrolysis and further hydroxylation and/or glucuronidation. After ester hydrolysis, FDU-PB-22 and FUB-PB-22 yielded the same metabolite M7, fluorobenzylindole-3-carboxylic acid (FBI-COOH). M7 and M6 (hydroxylated FBI-COOH) were the major metabolites. In authentic urine specimens after ß-glucuronidase hydrolysis, M6 and M7 also were the predominant metabolites. Based on our study, we recommend M6 (hydroxylated FBI-COOH) and M7 (FBI-COOH) as suitable urinary markers for documenting FDU-PB-22 and/or FUB-PB-22 intake.