Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy.

BACKGROUND: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. METHODS: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. RESULTS: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. CONCLUSIONS: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

The Casein kinase 1α agonist pyrvinium attenuates Wnt-mediated CK1α degradation via interaction with the E3 ubiquitin ligase component Cereblon.

The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN-CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.

Discovery, synthesis and characterization of a series of 7-aryl-imidazo[1,2-a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors.

The activin-like kinases are a family of kinases that play important roles in a variety of disease states. Of this class of kinases, ALK2, has been shown by a gain-of-function to be the primary driver of the childhood skeletal disease fibrodysplasia ossificans progressiva (FOP) and more recently the pediatric cancer diffuse intrinsic pontine glioma (DIPG). Herein, we report our efforts to identify a novel imidazo[1,2-a]pyridine scaffold as potent inhibitors of ALK2 with good in vivo pharmacokinetic properties suitable for future animal studies.

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

RATIONALE: The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. OBJECTIVE: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. METHODS AND RESULTS: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). CONCLUSIONS: The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.

Hypoplastic Left Heart Syndrome Sequencing Reveals a Novel NOTCH1 Mutation in a Family with Single Ventricle Defects.

Hypoplastic left heart syndrome (HLHS) has been associated with germline mutations in 12 candidate genes and a recurrent somatic mutation in HAND1 gene. Using targeted and whole exome sequencing (WES) of heart tissue samples from HLHS patients, we sought to estimate the prevalence of somatic and germline mutations associated with HLHS. We performed Sanger sequencing of the HAND1 gene on 14 ventricular (9 LV and 5 RV) samples obtained from HLHS patients, and WES of 4 LV, 2 aortic, and 4 matched PBMC samples, analyzing for sequence discrepancy. We also screened for mutations in the 12 candidate genes implicated in HLHS. We found no somatic mutations in our HLHS cohort. However, we detected a novel germline frameshift/stop-gain mutation in NOTCH1 in a HLHS patient with a family history of both HLHS and hypoplastic right heart syndrome (HRHS). Our study, involving one of the first familial cases of single ventricle defects linked to a specific mutation, strengthens the association of NOTCH1 mutations with HLHS and suggests that the two morphologically distinct single ventricle conditions, HLHS and HRHS, may share a common molecular and cellular etiology. Finally, somatic mutations in the LV are an unlikely contributor to HLHS.

Inhibition of GPR68 kills glioblastoma in zebrafish xenograft models.

OBJECTIVE: Inhibition and knockdown of GPR68 negatively affects glioblastoma cell survival in vitro by inducing ferroptosis. Herein, we aimed to demonstrate that inhibition of GPR68 reduces the survival of glioblastoma cells in vivo using two orthotopic larval xenograft models in Danio rerio, using GBM cell lines U87-MG and U138-MG. In vivo survival of the cancer cells was assessed in the setting of GPR68 inhibition or knockdown. RESULTS: In vitro, shRNA-mediated knockdown of GPR68 inhibition demonstrated potent cytotoxic effects against U87 and U138 glioblastoma cell lines. This effect was associated with increased intracellular lipid peroxidation, suggesting ferroptosis as the underlying mechanism of cell death. Translating these findings in vivo, we established a novel xenograft model in zebrafish by successfully grafting fluorescently labeled human glioblastoma cells, which were previously shown to overexpress GPR68. shRNA knockdown of GPR68 significantly reduced the viability of grafted GBM cells within this model. Additionally, treatment with ogremorphin (OGM), a highly specific small molecule inhibitor of GPR68, also reduced the viability of grafted GBM cells with limited toxicity to the developing zebrafish embryos. This study suggests that therapeutic targeting of GPR68 with small molecules like OGM represents a promising approach for the treatment of GBM.

Simulating the Motion Underlying the Mechanism of Thioredoxin Reductase.

Thioredoxin reductase (TrxR) is an essential antioxidant in most cells; it reduces thioredoxin (Trx) and several more substrates, utilizing NADPH. However, the enzyme's internal active site is too small to accommodate the Trx substrate. Thus, TrxR evolved a disulfide shuttle that can carry reducing equivalents from the active site to the docking site of thioredoxin on the enzyme surface. Yet, in all available atomic structures of TrxR, access to the active site by the shuttle is sterically blocked. We find with computational dynamics that thermal motion at 37 °C allows the oxidized shuttle x to transiently access the active site. Once the shuttle is reduced, it becomes polar. Again, with molecular dynamics, we show that the polar shuttle will move outward toward the solution interface, whereas the oxidized, neutral shuttle will not. This work provides physical evidence for crucial steps in the enzyme mechanism that thus far were just conjectures. The total shuttle motion, from the active site toward the surface, is over 20 Å. TrxR may thus also be termed a molecular machine.

GPR68-ATF4 signaling is a novel prosurvival pathway in glioblastoma activated by acidic extracellular microenvironment.

BACKGROUND: Glioblastoma multiforme (GBM) stands as a formidable challenge in oncology because of its aggressive nature and severely limited treatment options. Despite decades of research, the survival rates for GBM remain effectively stagnant. A defining hallmark of GBM is a highly acidic tumor microenvironment, which is thought to activate pro-tumorigenic pathways. This acidification is the result of altered tumor metabolism favoring aerobic glycolysis, a phenomenon known as the Warburg effect. Low extracellular pH confers radioresistant tumors to glial cells. Notably GPR68, an acid sensing GPCR, is upregulated in radioresistant GBM. Usage of Lorazepam, which has off target agonism of GPR68, is linked to worse clinical outcomes for a variety of cancers. However, the role of tumor microenvironment acidification in GPR68 activation has not been assessed in cancer. Here we interrogate the role of GPR68 specifically in GBM cells using a novel highly specific small molecule inhibitor of GPR68 named Ogremorphin (OGM) to induce the iron mediated cell death pathway: ferroptosis. METHOD: OGM was identified in a non-biased zebrafish embryonic development screen and validated with Morpholino and CRISPR based approaches. Next, A GPI-anchored pH reporter, pHluorin2, was stably expressed in U87 glioblastoma cells to probe extracellular acidification. Cell survival assays, via nuclei counting and cell titer glo, were used to demonstrate sensitivity to GPR68 inhibition in twelve immortalized and PDX GBM lines. To determine GPR68 inhibition's mechanism of cell death we use DAVID pathway analysis of RNAseq. Our major indication, ferroptosis, was then confirmed by western blotting and qRT-PCR of reporter genes including TFRC. This finding was further validated by transmission electron microscopy and liperfluo staining to assess lipid peroxidation. Lastly, we use siRNA and CRISPRi to demonstrate the critical role of ATF4 suppression via GPR68 for GBM survival. RESULTS: We used a pHLourin2 probe to demonstrate how glioblastoma cells acidify their microenvironment to activate the commonly over expressed acid sensing GPCR, GPR68. Using our small molecule inhibitor OGM and genetic means, we show that blocking GPR68 signaling results in robust cell death in all thirteen glioblastoma cell lines tested, irrespective of genetic and phenotypic heterogeneity, or resistance to the mainstay GBM chemotherapeutic temozolomide. We use U87 and U138 glioblastoma cell lines to show how selective induction of ferroptosis occurs in an ATF4-dependent manner. Importantly, OGM was not-acutely toxic to zebrafish and its inhibitory effects were found to spare non-malignant neural cells. CONCLUSION: These results indicate GPR68 emerges as a critical sensor for an autocrine pro-tumorigenic signaling cascade triggered by extracellular acidification in glioblastoma cells. In this context, GPR68 suppresses ATF4, inhibition of GPR68 increases expression of ATF4 which leads to ferroptotic cell death. These findings provide a promising therapeutic approach to selectively induce ferroptosis in glioblastoma cells while sparing healthy neural tissue.

Assessment of the therapeutic efficacy of artemether-lumefantrine in the treatment of uncomplicated Plasmodium falciparum malaria in northern KwaZulu-Natal: an observational cohort study.

BACKGROUND: Recent malaria epidemics in KwaZulu-Natal indicate that effective anti-malarial therapy is essential for malaria control. Although artemether-lumefantrine has been used as first-line treatment for uncomplicated Plasmodium falciparum malaria in northern KwaZulu-Natal since 2001, its efficacy has not been assessed since 2002. The objectives of this study were to quantify the proportion of patients treated for uncomplicated P. falciparum malaria with artemether-lumefantrine who failed treatment after 28 days, and to determine the prevalence of molecular markers associated with artemether-lumefantrine and chloroquine resistance. METHODS: An observational cohort of 49 symptomatic patients, diagnosed with uncomplicated P. falciparum malaria by rapid diagnostic test, had blood taken for malaria blood films and P. falciparum DNA polymerase chain reaction (PCR). Following diagnosis, patients were treated with artemether-lumefantrine (Coartem®) and invited to return to the health facility after 28 days for repeat blood film and PCR. All PCR P. falciparum positive samples were analysed for molecular markers of lumefantrine and chloroquine resistance. RESULTS: Of 49 patients recruited on the basis of a positive rapid diagnostic test, only 16 were confirmed to have P. falciparum by PCR. At follow-up, 14 were PCR-negative for malaria, one was lost to follow-up and one blood specimen had insufficient blood for a PCR analysis. All 16 with PCR-confirmed malaria carried a single copy of the multi-drug resistant (mdr1) gene, and the wild type asparagine allele mdr1 codon 86 (mdr1 86N). Ten of the 16 samples carried the wild type haplotype (CVMNK) at codons 72-76 of the chloroquine resistance transporter gene (pfcrt); three samples carried the resistant CVIET allele; one carried both the resistant and wild type, and in two samples the allele could not be analysed. CONCLUSIONS: The absence of mdr1 gene copy number variation detected in this study suggests lumefantrine resistance has yet to emerge in KwaZulu-Natal. In addition, data from this investigation implies the possible re-emergence of chloroquine-sensitive parasites. Results from this study must be viewed with caution, given the extremely small sample size. A larger study is needed to accurately determine therapeutic efficacy of artemether-lumefantrine and resistance marker prevalence. The high proportion of rapid diagnostic test false-positive results requires further investigation.

Identifying genetic variants associated with the ICD10 (International Classification of Diseases10)-based diagnosis of cerebrovascular disease using a large-scale biomedical database.

OBJECTIVES: To utilize the UK Biobank to identify genetic variants associated with the ICD10 (International Classification of Diseases10)-based diagnosis of cerebrovascular disease (CeVD). BACKGROUND: Cerebrovascular disease occurs because of a complex interplay between vascular, environmental, and genetic factors. It is the second leading cause of disability worldwide. Understanding who may be genetically predisposed to cerebrovascular disease can help guide preventative efforts. Moreover, there is considerable interest in the use of real-world data, such as EHR (electronic health records) to better understand disease mechanisms and to discover new treatment strategies, but whether ICD10-based diagnosis can be used to study CeVD genetics is unknown. METHODS: Using the UK Biobank, we conducted a genome-wide association study (GWAS) where we analyzed the genomes of 11,155 cases and 122,705 controls who were sex, age and ancestry-matched in a 1:11 case: control design. Genetic variants were identified by Plink's firth logistic regression and assessed for association with the ICD10 codes corresponding to CeVD. RESULTS: We identified two groups of SNPs closely linked to PITX2 and LRRTM4 that were significantly associated with CeVD in this study (p < 5 x 10-8) and had a minor allele frequency of > 0.5%. DISCUSSION: Disease assignment based on ICD10 codes may underestimate prevalence; however, for CeVD, this does not appear to be the case. Compared to the age- and sex-matched control population, individuals with CeVD were more frequently diagnosed with comorbid conditions, such as hypertension, hyperlipidemia & atrial fibrillation or flutter, confirming their contribution to CeVD. The UK Biobank based ICD10 study identified 2 groups of variants that were associated with CeVD. The association between PITX2 and CeVD is likely explained by the increased rates of atrial fibrillation and flutter. While the mechanism explaining the relationship between LRRTM4 and CeVD is unclear, this has been documented in previous studies.

Identification of Novel Genetic Variants and Comorbidities Associated With ICD-10-Based Diagnosis of Hypertrophic Cardiomyopathy Using the UK Biobank Cohort.

Objectives: To identify previously unrecognized genetic variants and clinical variables associated with the ICD-10 (International Classification of Diseases 10)-based diagnosis of hypertrophic cardiomyopathy in the UK Biobank cohort. Background: Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disorder with more than 2000 known mutations in one of eight genes encoding sarcomeric proteins. However, there is considerable variation in disease manifestation, suggesting the role of additional unrecognized contributors, genetic and otherwise. There is substantial interest in the use of real-world data, such as electronic health records to better understand disease mechanisms and discover new treatment strategies, but whether ICD-10-based diagnosis can be used to study HCM genetics is unknown. Methods: In a genome-wide association study (GWAS) using the UK Biobank, we analyzed the genomes of 363 individuals diagnosed with HCM based on ICD-10 coding compared to 7,260 age, ancestry, and sex-matched controls in a 1:20 case:control design. Genetic variants were analyzed by Plink's firth logistic regression and assessed for association with HCM. We also examined 61 biomarkers and other diagnoses in the 363 HCM cases and matched controls. Results: The prevalence of ICD-10-based diagnosis of HCM in the UK Biobank cohort was 1 in 1,342, suggesting disease assignment based on the two ICD-10 codes underestimates HCM prevalence. In addition, common cardiovascular comorbidities were more prevalent in ICD-10-based HCM cases in comparison to controls. We identified two novel, non-sarcomeric genetic variants in KMT2C rs78630626, and PARD3B rs188937806 that were associated with ICD-10 codes for HCM with genome-wide significance (p < 5 x 10-8). These are associated with an increased odds ratio (OR) of ∼3.8 for being diagnosed with HCM. Minor allele frequency (MAF) of each variant was >1%. Discussion: Disease assignment based strictly on ICD-10 codes may underestimate HCM prevalence. Individuals with HCM were more frequently diagnosed with several comorbid conditions, such as hypertension, atherosclerotic heart disease, diabetes, and kidney failure, suggesting they may contribute to disease manifestation. This UK Biobank database-based GWAS identified common variants in KMT2C and PARD3B that are associated with HCM diagnosis, which may represent novel modifier genes. Our study demonstrates the feasibility and limitations of conducting phenotypic and genotypic characterization of HCM based on ICD-10 diagnosis in a large population-based cohort.

Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni.

Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596'/Sec-597' pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596'/Sec-597' thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx deglutathionylation activity, whereas Cys-31 variants retained partial Grx deglutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the lags. A Sec-597 variant was still able to reduce a variety of substrates, albeit slowly, showing that selenocysteine is important but is not the sole determinant for the broad substrate tolerance of the enzyme. Our data show that Cys-520 and Cys-574 are not likely to be involved in the catalytic mechanism.

Multi-step usage of in vivo models during rational drug design and discovery.

In this article we propose a systematic development method for rational drug design while reviewing paradigms in industry, emerging techniques and technologies in the field. Although the process of drug development today has been accelerated by emergence of computational methodologies, it is a herculean challenge requiring exorbitant resources; and often fails to yield clinically viable results. The current paradigm of target based drug design is often misguided and tends to yield compounds that have poor absorption, distribution, metabolism, and excretion, toxicology (ADMET) properties. Therefore, an in vivo organism based approach allowing for a multidisciplinary inquiry into potent and selective molecules is an excellent place to begin rational drug design. We will review how organisms like the zebrafish and Caenorhabditis elegans can not only be starting points, but can be used at various steps of the drug development process from target identification to pre-clinical trial models. This systems biology based approach paired with the power of computational biology; genetics and developmental biology provide a methodological framework to avoid the pitfalls of traditional target based drug design.

Identification of novel genetic susceptibility loci for thoracic and abdominal aortic aneurysms via genome-wide association study using the UK Biobank Cohort.

BACKGROUND: Thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) are known to have a strong genetic component. METHODS AND RESULTS: In a genome-wide association study (GWAS) using the UK Biobank, we analyzed the genomes of 1,363 individuals with AAA compared to 27,260 age, ancestry, and sex-matched controls (1:20 case:control study design). A similar analysis was repeated for 435 individuals with TAA compared to 8,700 controls. Polymorphism with minor allele frequency (MAF) >0.5% were evaluated. We identified novel loci near LINC01021, ATOH8 and JAK2 genes that achieved genome-wide significance for AAA (p-value <5x10-8), in addition to three known loci. For TAA, three novel loci in CTNNA3, FRMD6 and MBP achieved genome-wide significance. There was no overlap in the genes associated with AAAs and TAAs. Additionally, we identified a linkage group of high-frequency variants (MAFs ~10%) encompassing FBN1, the causal gene for Marfan syndrome, which was associated with TAA. In FinnGen PheWeb, this FBN1 haplotype was associated with aortic dissection. Finally, we found that baseline bradycardia was associated with TAA, but not AAA. CONCLUSIONS: Our GWAS found that AAA and TAA were associated with distinct sets of genes, suggesting distinct underlying genetic architecture. We also found association between baseline bradycardia and TAA. These findings, including JAK2 association, offer plausible mechanistic and therapeutic insights. We also found a common FBN1 linkage group that is associated with TAA and aortic dissection in patients who do not have Marfan syndrome. These FBN1 variants suggest shared pathophysiology between Marfan disease and sporadic TAA.

The E3 ubiquitin ligase component, Cereblon, is an evolutionarily conserved regulator of Wnt signaling.

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.

PEGylated PLGA Nanoparticle Delivery of Eggmanone for T Cell Modulation: Applications in Rheumatic Autoimmunity.

BACKGROUND: Helper T cell activity is dysregulated in a number of diseases including those associated with rheumatic autoimmunity. Treatment options are limited and usually consist of systemic immune suppression, resulting in undesirable consequences from compromised immunity. Hedgehog (Hh) signaling has been implicated in the activation of T cells and the formation of the immune synapse, but remains understudied in the context of autoimmunity. Modulation of Hh signaling has the potential to enable controlled immunosuppression but a potential therapy has not yet been developed to leverage this opportunity. METHODS: In this work, we developed biodegradable nanoparticles to enable targeted delivery of eggmanone (Egm), a specific Hh inhibitor, to CD4+ T cell subsets. We utilized two FDA-approved polymers, poly(lactic-co-glycolic acid) and polyethylene glycol, to generate hydrolytically degradable nanoparticles. Furthermore, we employed maleimide-thiol mediated conjugation chemistry to decorate nanoparticles with anti-CD4 F(ab') antibody fragments to enable targeted delivery of Egm. RESULTS: Our novel delivery system achieved a highly specific association with the majority of CD4+ T cells present among a complex cell population. Additionally, we have demonstrated antigen-specific inhibition of CD4+ T cell responses mediated by nanoparticle-formulated Egm. CONCLUSION: This work is the first characterization of Egm's immunomodulatory potential. Importantly, this study also suggests the potential benefit of a biodegradable delivery vehicle that is rationally designed for preferential interaction with a specific immune cell subtype for targeted modulation of Hh signaling.

Should we overcome the resistance to bioelectrical impedance in heart failure?

INTRODUCTION: Heart failure is associated with increased neurohormonal activation that results in changes in body composition including volume overload and the loss of skeletal muscle, body fat, and bone density. Bioelectrical impedance measures body composition based on the conduction of electrical current through body fluids. AREAS COVERED: The PubMed and Scopus databases were reviewed up to the third week of June 2020. Cross-sectional studies, retrospective observational studies, prospective observational studies, and randomized controlled trials have examined numerous bioelectrical impedance monitoring strategies to guide the diagnosis, prognosis, and treatment of heart failure. These monitoring strategies include intrathoracic impedance, lung impedance, bioelectrical impedance vector analysis, leg bioelectrical impedance, and thoracic bioreactance. EXPERT COMMENTARY: Based on the current evidence, more studies are needed to validate bioelectrical impedance in heart failure. Lung impedance appears to be useful for guiding heart failure treatment in patients with ST-elevation myocardial infarction and improving outcomes in outpatients with heart failure. Furthermore, bioelectrical impedance has potential as a noninvasive, quantitative heart failure variable for population-based research.

BMPing Up Healing Capacity with FKBP12 Ligand.

While the wound healing property of the macrolide FK506 is well known, the underlying mechanism has been elusive. In this issue of Cell Chemical Biology, Peiffer et al. (2019) utilize FKBP12 ligand to demonstrate that wound healing effects of FK506 occur via activation of the BMP (bone morphogenic protein) signaling pathway.

Function of Glu-469' in the acid-base catalysis of thioredoxin reductase from Drosophila melanogaster.

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. Because dipteran insects such as Drosophila melanogaster lack glutathione reductase, their TrxRs are particularly important for antioxidant protection; reduced Trx reacts nonenzymatically with oxidized glutathione to maintain a high glutathione/glutathione disulfide ratio. Like other members of the pyridine nucleotide-disulfide oxidoreductase family, TrxR is a homodimer; in the enzyme from D. melanogaster (DmTrxR), each catalytically active unit consists of three redox centers: FAD and an N-terminal Cys-57-Cys-62 redox-active disulfide from one monomer and a Cys-489'-Cys-490' C-terminal redox-active disulfide from the second monomer. A dyad of His-464' and Glu-469' in TrxR acts as the acid-base catalyst of the dithiol-disulfide interchange reactions required in catalysis [Huang, H.-H., et al. (2008) Biochemistry 47, 1721-1731]. In this investigation, the role of Glu-469' in catalysis by DmTrxR has been studied. The E469'A and E469'Q DmTrxR variants retain 28 and 35% of the wild-type activity, respectively, indicating that this glutamate residue is important but not critical to catalysis. The pH dependence of V(max) for both glutamate variants yields pK(a) values of 6.0 and 8.7, compared to those in the wild-type enzyme of 6.4 and 9.3, respectively, indicating that the basicity of His-464' in TrxR in complex with its substrate, DmTrx-2, is significantly lower in the glutamate variants than in wild-type enzyme. The rates of some steps in the reductive half-reactions in both glutamate variants are much slower than those of the wild-type enzyme. On the basis of our observations, it is proposed that the function of Glu-469' is to facilitate the positioning of His-464' toward the interchange thiol, Cys-57, as suggested for the analogous residue in glutathione reductase.