Mammalian HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated ...degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78 independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR via Insigs, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent E3 ligase, partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation.
This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor ...activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.
•Prevalence of FcRH5 expression in multiple myeloma is 100%•Anti-FcRH5/CD3 TDB redirects T cells to kill myeloma cells•Target clustering and CD45 exclusion activate T cells•Anti-FcRH5/CD3 TDB is a highly efficacious immunotherapy for myeloma
Li et al. report that the size and epitope location of the target play a key role in the efficiency of T cell activation induced by T cell-dependent bispecific antibodies (TDBs). They develop a TDB targeting FcRH5 expressed in all multiple myeloma tumor cells and show its potential in treating this disease.
The application of forward genetic screens to cultured human cells represents a powerful method to study gene function. The repurposing of the bacterial CRISPR/Cas9 system provides an effective ...method to disrupt gene function in mammalian cells, and has been applied to genome-wide screens. Here, we compare the efficacy of genome-wide CRISPR/Cas9-mediated forward genetic screens versus gene-trap mutagenesis screens in haploid human cells, which represent the existing 'gold standard' method. This head-to-head comparison aimed to identify genes required for the endoplasmic reticulum-associated degradation (ERAD) of MHC class I molecules. The two approaches show high concordance (>70%), successfully identifying the majority of the known components of the canonical glycoprotein ERAD pathway. Both screens also identify a role for the uncharacterized gene TXNDC11, which we show encodes an EDEM2/3-associated disulphide reductase. Genome-wide CRISPR/Cas9-mediated screens together with haploid genetic screens provide a powerful addition to the forward genetic toolbox.
Galectins are a family of lectin binding proteins expressed both intracellularly and extracellularly. Galectin-3 (Gal-3, also known as LGALS3) is expressed at the cell surface; however, Gal-3 lacks a ...signal sequence, and the mechanism of Gal-3 transport to the cell surface remains poorly understood. Here, using a genome-wide CRISPR/Cas9 forward genetic screen for regulators of Gal-3 cell surface localization, we identified genes encoding glycoproteins, enzymes involved in N-linked glycosylation, regulators of ER-Golgi trafficking and proteins involved in immunity. The results of this screening approach led us to address the controversial role of N-linked glycosylation in the transport of Gal-3 to the cell surface. We find that N-linked glycoprotein maturation is not required for Gal-3 transport from the cytosol to the extracellular space, but is important for cell surface binding. Additionally, secreted Gal-3 is predominantly free and not packaged into extracellular vesicles. These data support a secretion pathway independent of N-linked glycoproteins and extracellular vesicles.
A subset of TLRs is specialised in the detection of incoming pathogens by sampling endosomes for nucleic acid contents. Among them, TLR3 senses the abnormal presence of double-stranded RNA in the ...endosomes and initiates a potent innate immune response via activation of NF-κB and IRF3. Nevertheless, mechanisms governing TLR3 regulation remain poorly defined. To identify new molecular players involved in the TLR3 pathway, we performed a genome-wide screen using CRISPR/Cas9 technology. We generated TLR3+ reporter cells carrying a NF-κB-responsive promoter that controls GFP expression. Cells were next transduced with a single-guide RNA (sgRNA) library, subjected to sequential rounds of stimulation with poly(I:C) and sorting of the GFP-negative cells. Enrichments in sgRNA estimated by deep sequencing identified genes required for TLR3-induced activation of NF-κB. Among the hits, five genes known to be critically involved in the TLR3 pathway, including TLR3 itself and the chaperone UNC93B1, were identified by the screen, thus validating our strategy. We further studied the top 40 hits and focused on the transcription factor aryl hydrocarbon receptor (AhR). Depletion of AhR had a dual effect on the TLR3 response, abrogating IL-8 production and enhancing IP-10 release. Moreover, in primary human macrophages exposed to poly(I:C), AhR activation enhanced IL-8 and diminished IP-10 release. Overall, these results reveal AhR plays a role in the TLR3 cellular innate immune response.
Correctly identifying animals that are truly infected with a pathogen using ante-mortem tests is the cornerstone of any disease eradication programme. Failure to identify all infected animals will ...impede the progress towards controlling and eradicating disease and may also have unforeseen consequences when specific prevention measures are in place to avoid animal-to-animal transmission. In the case of bovine tuberculosis (bTB), the screening ante-mortem test, the Single Comparative Intradermal Tuberculin Test (SCITT), can exhibit moderate sensitivity which can result in a “hidden burden” of infection residing within the population. Using an animal-level dataset relating to the disclosure of infected cattle with Mycobacterium bovis, the causative agent of bTB within infected herds in Northern Ireland, we investigated what factors influenced the probability of an animal being a false-negative when truly infected (using post-mortem (PM) microbiological culture confirmation results to assess infection status).
We found that different risk factors affected the probability of a test-negative outcome on infected animals depending on the ante-mortem test or their combination (SICTT and/or interferon gamma (IFN-ɣ) testing). Using multivariable models, SCITT disclosure performance varied significantly by age, location (region), and production type. The IFN-ɣ tests were significantly affected by region or season, but these effects depended on the cut-off used during interpretation of the test which affected the tests characteristics. Parallel use of SCITT and IFN-ɣ tests resulted in the least number of false-negatives, and their disclosure was affected by season and age-class.
Understanding the factors that lead to the non-disclosure of infected animals is essential to optimise large-scale bTB disease eradication programmes.
•Risk factors for cattle being disclosed as false-negatives were investigated.•False-negatives were defined by SCITT, IFN-ɣ tests and parallel use of these tests.•Disclosure varied significantly across test types.•Age, region, season and production type were risk factors for failure to disclose infection at the ante-mortem tests•Knowledge of risk factors affecting non-disclosure of infected animals is essential to disease control programmes
The objective of Table 2 was to examine whether certain regions were more likely to have positive samples than other sites. ...the results used for Table 2 were restricted to those badgers sampled ...more than five times. Region Sites sampled if possible Proportion of M bovis-positive (number of positive samples/numberof samples collected) Odds ratio (95%confidence interval) Abdomen Kidney, liver, mesenteric lymph node, spleen 0.05 (102/2022) 1 Carcase Prescapular and popliteal pool 0.09 (76/831) 1.89 (1.37 to 2.61) Head Masseter muscle, retropharyngeal lymph node, submandibular lymph node, tonsil 0.17 (1/6) 3.76 (0.08 to 34.02) Thorax Lung, mediastinal lymph node 0.62 (8/13) 29.94 (8.47 to 118.71) Other Abscess swab, faeces, other lymph nodes, muscle, other lesions, urine 0.05 (114/2341) 0.96 (0.73 to 1.28) Samples were taken if the tissue was not overly damaged. Data on cattle were extracted from the Animal and Public Health Information System. 11 For each 5-km zone, the number of M bovis-positive unique herds (defined as having one or more tuberculosis reactors (defined as positive to the single intradermal comparative cervical tuberculin test) for 12 months preceding and 12 months following the date the badger was collected) was calculated, as well as the number of unique herds tested during the time period. The results of the survey have guided decisions for cattle bTB control at the local and national level, for example, local herd breakdown investigations and biosecurity advice, 2 20 and have been used in the design of wildlife interventions and research. 21-23 Despite the limitations, RTA surveys, compared with other field methods, represent a relatively inexpensive and non-invasive method to estimate the prevalence of tuberculosis in badgers.
Tobacco smoking causes lung cancer
, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA
. The profound effects of tobacco on the genome of ...lung cancer cells are well-documented
, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0-6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.
Psychedelic therapies can engender enduring improvements in psychological well-being. However, relatively little is known about the psychological mechanisms through which the salutary effects of ...psychedelics emerge. Through integrating extant research on psychedelics with contemporary existential psychology, we present a novel hypothesis that reduced death anxiety may be a key mechanism underpinning the therapeutic effects of psychedelics. In developing this hypothesis, we also provide a complementary review of mechanisms through which psychedelics may reduce death anxiety. We conclude that an awareness of the role of death anxiety in psychopathology has the potential to guide future research into psychedelic therapies.
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