Plasticity of the Cullin-RING Ligase Repertoire Shapes Sensitivity to Ligand-Induced Protein Degradation.

Inducing protein degradation via small molecules is a transformative therapeutic paradigm. Although structural requirements of target degradation are emerging, mechanisms determining the cellular response to small-molecule degraders remain poorly understood. To systematically delineate effectors required for targeted protein degradation, we applied genome-scale CRISPR/Cas9 screens for five drugs that hijack different substrate receptors (SRs) of cullin RING ligases (CRLs) to induce target proteolysis. We found that sensitivity to small-molecule degraders is dictated by shared and drug-specific modulator networks, including the COP9 signalosome and the SR exchange factor CAND1. Genetic or pharmacologic perturbation of these effectors impairs CRL plasticity and arrests a wide array of ligases in a constitutively active state. Resulting defects in CRL decommissioning prompt widespread CRL auto-degradation that confers resistance to multiple degraders. Collectively, our study informs on regulation and architecture of CRLs amenable for targeted protein degradation and outlines biomarkers and putative resistance mechanisms for upcoming clinical investigation.

Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders.

Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined 'functional hotspots'. Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.

BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment.

Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.

E3-Specific Degrader Discovery by Dynamic Tracing of Substrate Receptor Abundance.

Targeted protein degradation (TPD) is a new pharmacology based on small-molecule degraders that induce proximity between a protein of interest (POI) and an E3 ubiquitin ligase. Of the approximately 600 E3s encoded in the human genome, only around 2% can be co-opted with degraders. This underrepresentation is caused by a paucity of discovery approaches to identify degraders for defined E3s. This hampers a rational expansion of the druggable proteome and stymies critical advancements in the field, such as tissue- and cell-specific degradation. Here, we focus on dynamic NEDD8 conjugation, a post-translational, regulatory circuit that controls the activity of 250 cullin RING E3 ligases (CRLs). Leveraging this regulatory layer enabled us to develop a scalable assay to identify compounds that alter the interactome of an E3 of interest by tracing their abundance after pharmacologically induced auto-degradation. Initial validation studies are performed for CRBN and VHL, but proteomics studies indicate broad applicability for many CRLs. Among amenable ligases, we select CRLDCAF15 for a proof-of-concept screen, leading to the identification of a novel DCAF15-dependent molecular glue degrader inducing the degradation of RBM23 and RBM39. Together, this strategy empowers the scalable identification of degraders specific to a ligase of interest.

Integrated experimental-computational analysis of a HepaRG liver-islet microphysiological system for human-centric diabetes research.

Microphysiological systems (MPS) are powerful tools for emulating human physiology and replicating disease progression in vitro. MPS could be better predictors of human outcome than current animal models, but mechanistic interpretation and in vivo extrapolation of the experimental results remain significant challenges. Here, we address these challenges using an integrated experimental-computational approach. This approach allows for in silico representation and predictions of glucose metabolism in a previously reported MPS with two organ compartments (liver and pancreas) connected in a closed loop with circulating medium. We developed a computational model describing glucose metabolism over 15 days of culture in the MPS. The model was calibrated on an experiment-specific basis using data from seven experiments, where HepaRG single-liver or liver-islet cultures were exposed to both normal and hyperglycemic conditions resembling high blood glucose levels in diabetes. The calibrated models reproduced the fast (i.e. hourly) variations in glucose and insulin observed in the MPS experiments, as well as the long-term (i.e. over weeks) decline in both glucose tolerance and insulin secretion. We also investigated the behaviour of the system under hypoglycemia by simulating this condition in silico, and the model could correctly predict the glucose and insulin responses measured in new MPS experiments. Last, we used the computational model to translate the experimental results to humans, showing good agreement with published data of the glucose response to a meal in healthy subjects. The integrated experimental-computational framework opens new avenues for future investigations toward disease mechanisms and the development of new therapies for metabolic disorders.

Rational discovery of molecular glue degraders via scalable chemical profiling.

Targeted protein degradation is a new therapeutic modality based on drugs that destabilize proteins by inducing their proximity to E3 ubiquitin ligases. Of particular interest are molecular glues that can degrade otherwise unligandable proteins by orchestrating direct interactions between target and ligase. However, their discovery has so far been serendipitous, thus hampering broad translational efforts. Here, we describe a scalable strategy toward glue degrader discovery that is based on chemical screening in hyponeddylated cells coupled to a multi-omics target deconvolution campaign. This approach led us to identify compounds that induce ubiquitination and degradation of cyclin K by prompting an interaction of CDK12-cyclin K with a CRL4B ligase complex. Notably, this interaction is independent of a dedicated substrate receptor, thus functionally segregating this mechanism from all described degraders. Collectively, our data outline a versatile and broadly applicable strategy to identify degraders with nonobvious mechanisms and thus empower future drug discovery efforts.

[Cirrhosis and coagulation : implications for clinical practice]. / Cirrhose et coagulation : implications pour la pratique clinique.

Coagulation disorders related to abnormalities in hepatic synthesis are well known as prognostic factors in hepatic cirrhosis. The risk of bleeding, mainly linked to portal hypertension, must be weighed against the risk of thrombosis, the most frequent manifestation of which is portal venous thrombosis. Conventional laboratory tests are not a reliable reflection of this delicate balance. The use of prophylactic anticoagulation in hospitalized patients with decompensated hepatic cirrhosis or therapeutic anticoagulation in venous thrombosis is recommended in most cases, in the absence of contraindications.

Les troubles de la coagulation liés aux anomalies de la synthèse hépatique sont bien connus comme facteurs pronostiques de la cirrhose hépatique. Le risque hémorragique, principalement lié à l'hypertension portale, est à mettre en balance avec le risque thrombotique, dont la manifestation la plus fréquente est la thrombose veineuse porte. Les tests de laboratoire classiques ne sont pas un reflet fiable de cet équilibre fragile. L'utilisation d'une anticoagulation prophylactique chez les patients hospitalisés avec cirrhose hépatique décompensée ou d'une anticoagulation thérapeutique en cas de thrombose veineuse est recommandée dans la plupart des cas, en l'absence de contre-indications.

Changes of Protein Turnover in Aging Caenorhabditis elegans.

Protein turnover rates severely decline in aging organisms, including C. elegans However, limited information is available on turnover dynamics at the individual protein level during aging. We followed changes in protein turnover at one-day resolution using a multiple-pulse 15N-labeling and accurate mass spectrometry approach. Forty percent of the proteome shows gradual slowdown in turnover with age, whereas only few proteins show increased turnover. Decrease in protein turnover was consistent for only a minority of functionally related protein subsets, including tubulins and vitellogenins, whereas randomly diverging turnover patterns with age were the norm. Our data suggests increased heterogeneity of protein turnover of the translation machinery, whereas protein turnover of ubiquitin-proteasome and antioxidant systems are well-preserved over time. Hence, we presume that maintenance of quality control mechanisms is a protective strategy in aging worms, although the ultimate proteome collapse is inescapable.

Morphological characteristics of osteotomies using different piezosurgical devices. A scanning electron microscopic evaluation.

PURPOSE: The objective of this study was to compare morphological characteristics of osteotomies performed by 6 Piezosurgical devices. MATERIALS AND METHODS: The 6 Piezosurgical units were: (a) Piezotom, (b) SurgySonic, (c) Piezon Master Surgery, (d) VarioSurg, (e) Surgybone, and (f) Piezosurgery 3. Osteotomies on 9 freshly slaughtered cattle ribs (2 cuts by each unit, per rib) from the cortical (first cut at 5 mm) to the cancellous (second cut at 3 mm) bone layer were performed. The osteotomy margins were compared using scanning electron microscopy analysis. The cutting areas, osteotomy bottoms, and osteotomy margins were analyzed morphologically. Statistical evaluation of the 2 cuts regarding the design of the tips (a-d: tapered tool shanks, f: parallel tool shank) was performed by an unpaired t test. RESULTS: Morphological characteristics were different for each Piezosurgical unit and each examined area. A significant difference (P = 0.0209) of the upper width of the first cut between tapered and parallel tips was shown. CONCLUSIONS: The morphological characteristics of the produced Piezosurgical osteotomies vary and depend on the Piezosurgical unit and tip.

A microfluidic thyroid-liver platform to assess chemical safety in humans.

Thyroid hormones (THs) are crucial regulators of human metabolism and early development. During the safety assessment of plant protection products, the human relevance of chemically induced TH perturbations observed in test animals remains uncertain. European regulatory authorities request follow-up in vitro studies to elucidate human-relevant interferences on thyroid gland function or TH catabolism through hepatic enzyme induction. However, human in vitro assays based on single molecular initiating events poorly reflect the complex TH biology and related liver-thyroid axis. To address this complexity, we present human three-dimensional thyroid and liver organoids with key functions of TH metabolism. The thyroid model resembles in vivo-like follicular architecture and a TSH-dependent triiodothyronine synthesis over 21 days, which is inhibited by methimazole. The HepaRG-based liver model, secreting the critical TH-binding proteins albumin and thyroxine-binding globulin, emulates an active TH catabolism via the formation of glucuronidated and sulfated thyroxine (gT4/sT4). Activation of the nuclear receptors PXR and AHR was demonstrated via the induction of specific CYP isoenzymes by rifampicin, pregnenolone-16α-carbonitrile, and ß-naphthoflavone. However, this nuclear receptor activation, assumed to regulate UDP-glucuronosyltransferases and sulfotransferases, appeared to have no effect on gT4 and sT4 formation in this human-derived hepatic cell line model. Finally, established single-tissue models were successfully co-cultured in a perfused two-organ chip for 21 days. In conclusion, this model presents a first step towards a complex multimodular human platform that will help to identify both direct and indirect thyroid disruptors that are relevant from a human safety perspective.

A rodent thyroid-liver chip to capture thyroid toxicity on organ function level.

Endocrine disruption by environmental chemicals continues to be a concern for human safety. The rat, a widely used model organism in toxicology, is very sensitive to chemical-induced thyroid perturbation, e.g., histopathological alterations in thyroid tissue. Species differences in the susceptibility to thyroid perturbation lead to uncertainty in human safety risk assessments. Hazard identification and characterization of chemically induced thyroid perturbation would therefore benefit from in vitro models addressing different mechanisms of action in a single functional assay, ideally across species. We here introduce a rat thyroid-liver chip that enables simultaneous identification of direct and indirect (liver-mediated) thyroid perturbation on organ-level functions in vitro. A second manuscript describes our work toward a human thyroid-liver chip (Kühnlenz et al., 2022). The presented microfluidic model consisting of primary rat thyroid follicles and liver 3D spheroids maintains a tissue-specific phenotype for up to 21 days. More precisely, the thyroid model exhibits a follicular architecture expressing basolateral and apical markers and secretes T4. Likewise, liver spheroids retain hepatocellular characteristics, e.g., a stable release of albumin and urea, the presence of bile canalicular networks, and the formation of T4-glucuronide. Experiments with reference chemicals demonstrated proficiency to detect direct and indirect mechanisms of thyroid perturbation through decreased thyroid hormone secretion and increased gT4 formation, respectively. Prospectively this rat thyroid-liver chip model, together with its human counterpart, may support a species-specific quantitative in vitro to in vivo extrapolation to improve a data-driven and evidence-based human safety risk assessment with significant contributions to the 3R principles.

Towards safety4.0: A novel approach for flexible human-robot-interaction based on safety-related dynamic finite-state machine with multilayer operation modes.

In the era of Industry 4.0 and agile manufacturing, the conventional methodologies for risk assessment, risk reduction, and safety procedures may not fulfill the End-User requirements, especially the SMEs with their product diversity and changeable production lines and processes. This work proposes a novel approach for planning and implementing safe and flexible Human-Robot-Interaction (HRI) workspaces using multilayer HRI operation modes. The collaborative operation modes are grouped in different clusters and categorized at various levels systematically. In addition to that, this work proposes a safety-related finite-state machine for describing the transitions between these modes dynamically and properly. The proposed approach is integrated into a new dynamic risk assessment tool as a promising solution toward a new safety horizon in line with industry 4.0.

Influence of sociodemographic factors on Helicobacter pylori prevalence variability among schoolchildren in Leipzig, Germany. A long-term follow-up study.

BACKGROUND: Until the beginning of this decade the assumption was that the Helicobacter pylori prevalence increases with the age of the population under consideration. More and more epidemiological studies have been suggestive of constancy in Helicobacter pylori prevalence, but to date there has been no long-term follow-up study in a large group of children confirming this hypothesis. METHODS: Following up our study of H. pylori among school starters and 2nd graders in 1998 and 2000, we conducted a third phase of this study in 2006 using the [13C]-Urea Breath Test and a detailed parent-completed epidemiological questionnaire to evaluate the development of prevalence among the overall population of 8th graders in the city of Leipzig (n = 1,905), and especially of the subgroup of participants who took part in all three study phases (n = 751). RESULTS: The overall H. pylori prevalence was 6.5% and had not significantly changed since 1998 and 2000 (6.1%, 5.7% respectively). However, we noticed a significant lower prevalence in the subgroup that participated in all study phases (2.7%). Moreover, we observed a dependence of prevalence on distribution of sociodemographic risk factors such as foreign nationality of at least one parent, birth outside of Germany, low parental education and unemployment, and > or = 2 older siblings. CONCLUSION: The variability in prevalence is most likely a result of unequal sociodemographic family backgrounds.

[Role of C-reactive protein in the diagnosis, prognosis and follow-up of community-acquired pneumonia]. / Utilité du dosage de la CRP dans le diagnostic, le pronostic et le suivi de la pneumonie acquise dans la communauté.

Community-acquired pneumonia (CAP) is a major clinical problem in terms of morbidity, mortality, and use of hospital resources. It is well recognized that a delay in making the diagnosis and instituting appropriate antibiotic treatment is associated with an increased mortality. C-reactive protein may be helpful in the management of patients with CAP. CRP is widely used in the management of CAP, including diagnosis, prognosis and follow-up. But its usefulness is not known. The aim of this systematic review was to evaluate the usefulness of CRP in the diagnosis, prognosis and follow-up of CAP.

Homolog-Selective Degradation as a Strategy to Probe the Function of CDK6 in AML.

The design of selective small molecules is often stymied by similar ligand binding pockets. Here, we report BSJ-03-123, a phthalimide-based degrader that exploits protein-interface determinants to achieve proteome-wide selectivity for the degradation of cyclin-dependent kinase 6 (CDK6). Pharmacologic CDK6 degradation targets a selective dependency of acute myeloid leukemia cells, and transcriptomics and phosphoproteomics profiling of acute degradation of CDK6 enabled dynamic mapping of its immediate role in coordinating signaling and transcription.

Publisher Correction: Functional coupling of human pancreatic islets and liver spheroids on-a-chip: Towards a novel human ex vivo type 2 diabetes model.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Field performance of the Determine HBsAg point-of-care test for diagnosis of hepatitis B virus co-infection among HIV patients in Zambia.

BACKGROUND: We evaluated the field performance of a rapid point-of-care (POC) test for hepatitis B surface antigen (HBsAg) that could support decentralization and scale-up of hepatitis B virus (HBV) diagnosis in Africa. OBJECTIVE: To determine the field performance of the Determine HBsAg POC test for diagnosis of HBV co-infection among HIV patients in Zambia. STUDY DESIGN: Between 2013-2014, we screened HIV-infected adults for HBsAg at two urban clinics in Zambia. A subset were tested with the POC Determine HBsAg (Alere, USA) by finger prick in the clinic and HBsAg serology (Access2Analyzer, Beckman Coulter) at a reference laboratory. If either test was reactive, we determined HBV viral load (VL) and genotype. We described patient demographic and clinical characteristics (including liver fibrosis) and assessed the sensitivity, specificity, positive and negative predictive values (PPV and NPV) of the Determine test. In secondary analyses, we assessed sensitivity among patients with replicating HBV (i.e., VL>20 IU/ml) and with high HBV VL (i.e.,>20,000IU/ml). RESULTS: Among 412 participants with both HBsAg tests, median age was 34 years, 51% were women, and median CD4 was 208 cells/mm3. By serology, 66 (16%) were HBsAg-positive. Overall Determine had 87.9% sensitivity, 99.7% specificity, 98.3% PPV, and 97.7% NPV. Six of 8 patients with false negative results had undetectable HBV VL and no evidence of significant liver fibrosis. Test sensitivity was 95.9% among the 51 with replicating HBV and 100% among the 28 with high HBV VL. CONCLUSIONS: Determine HBsAg is a cheaper alternative HBV testing option compared to the gold standard ELISA and has high specificity and good sensitivity in the field among HIV-infected individuals.

Coacervation-Mediated Combinatorial Synthesis of Biomatrices for Stem Cell Culture and Directed Differentiation.

Combinatorial screening represents a promising strategy to discover biomaterials for tailored cell culture applications. Although libraries incorporating different biochemical cues have been investigated, few simultaneously recapitulate relevant biochemical, physical, and dynamic features of the extracellular matrix (ECM). Here, a noncovalent system based on liquid-liquid phase separation (coacervation) and gelation mediated by glycosaminoglycan (GAG)-peptide interactions is reported. Multiple biomaterial libraries are generated using combinations of sulfated glycosaminoglycans and poly(ethylene glycol)-conjugated peptides. Screening these biomaterials reveals preferred biomatrices for the attachment of six cell types, including primary mesenchymal stromal cells (MSCs) and primary neural precursor cells (NPCs). Incorporation of GAGs sustains the expansion of all tested cell types comparable to standard cell culture surfaces, while osteogenic differentiation of MSC and neuronal differentiation of NPC are promoted on chondroitin and heparan biomatrices, respectively. The presented noncovalent system provides a powerful tool for developing tissue-specific ECM mimics.

Translation Termination Factor GSPT1 Is a Phenotypically Relevant Off-Target of Heterobifunctional Phthalimide Degraders.

Protein degradation is an emerging therapeutic strategy with a unique molecular pharmacology that enables the disruption of all functions associated with a target. This is particularly relevant for proteins depending on molecular scaffolding, such as transcription factors or receptor tyrosine kinases (RTKs). To address tractability of multiple RTKs for chemical degradation by the E3 ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN), we synthesized a series of phthalimide degraders based on the promiscuous kinase inhibitors sunitinib and PHA665752. While both series failed to induce degradation of their consensus targets, individual molecules displayed pronounced efficacy in leukemia cell lines. Orthogonal target identification supported by molecular docking led us to identify the translation termination factor G1 to S phase transition 1 (GSPT1) as a converging off-target, resulting from inadvertent E3 ligase modulation. This research highlights the importance of monitoring degradation events that are independent of the respective targeting ligand as a unique feature of small-molecule degraders.