PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development

Insights into molecular mechanisms of disease in Neurodegeneration with Brain Iron Accumulation; unifying theories.

Neurodegeneration with brain iron accumulation (NBIA) is a group of disorders characterised by dystonia, parkinsonism and spasticity. Iron accumulates in the basal ganglia and may be accompanied by Lewy bodies, axonal swellings and hyperphosphorylated tau depending on NBIA subtype. Mutations in 10 genes have been associated with NBIA that include Ceruloplasmin (Cp) and Ferritin Light Chain (FTL), both directly involved in iron homeostasis, as well as Pantothenate Kinase 2 (PANK2), Phospholipase A2 group 6 (PLA2G6), Fatty acid hydroxylase 2 (FA2H), Coenzyme A synthase (COASY), C19orf12, WDR45 and DCAF17 (C2orf37). These genes are involved in seemingly unrelated cellular pathways, such as lipid metabolism, Coenzyme A synthesis and autophagy. A greater understanding of the cellular pathways that link these genes and the disease mechanisms leading to iron dyshomeostasis is needed. Additionally, the major overlap seen between NBIA and more common neurodegenerative diseases may highlight conserved disease processes. In this review, we will discuss clinical and pathological findings for each NBIA-related gene, discuss proposed disease mechanisms such as mitochondrial health, oxidative damage, autophagy/mitophagy and iron homeostasis and speculate potential overlap between NBIA subtypes

Gene co-expression networks shed light into diseases of brain iron accumulation

Aberrant brain iron deposition is observed in both common and rare neurodegenerative disorders, including those categorized as Neurodegeneration with Brain Iron Accumulation (NBIA), which are characterized by focal iron accumulation in the basal ganglia. Two NBIA genes are directly involved in iron metabolism, but whether other NBIA-related genes also regulate iron homeostasis in the human brain, and whether aberrant iron deposition contributes to neurodegenerative processes remains largely unknown. This study aims to expand our understanding of these iron overload diseases and identify relationships between known NBIA genes and their main interacting partners by using a systems biology approach. We used whole-transcriptome gene expression data from human brain samples originating from 101 neuropathologically normal individuals (10 brain regions) to generate weighted gene co-expression networks and cluster the 10 known NBIA genes in an unsupervised manner. We investigated NBIA-enriched networks for relevant cell types and pathways, and whether they are disrupted by iron loading in NBIA diseased tissue and in an in vivo mouse model. We identified two basal ganglia gene co-expression modules significantly enriched for NBIA genes, which resemble neuronal and oligodendrocytic signatures. These NBIA gene networks are enriched for iron-related genes, and implicate synapse and lipid metabolism related pathways. Our data also indicates that these networks are disrupted by excessive brain iron loading. We identified multiple cell types in the origin of NBIA disorders. We also found unforeseen links between NBIA networks and iron-related processes, and demonstrate convergent pathways connecting NBIAs and phenotypically overlapping diseases. Our results are of further relevance for these diseases by providing candidates for new causative genes and possible points for therapeutic intervention

Can BCAT1 Expression Level Help Predict Disease Progression in Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is the most common blood cancer in the UK, with an incidence of >3500 newly diagnosed cases per year resulting in >1000 deaths. Disease prevalence increases with age, where the majority of patients are >65 years old [1]. CLL is a largely indolent disease and is routinely staged according to the Binet system as follows; stage A (involving 0-2 lymphoid sites), stage B (involving 2-5 lymphoid cites) and stage C (platelets < 1x1011/L or haemoglobin <10g/dL), the latter stage reflecting loss of bone marrow function [2]. Whilst for some stage A patients, the disease may remain stable for many decades (median life expectancy of 13 years), for others the disease progresses more rapidly [3]. This observation likely reflects the genetic and molecular heterogeneity of CLL. As such there are several prognostic risk factors used to stratify newly diagnosed patients, which include; trisomy-12, 13q/17p/11q23 deletion, advanced stage, males>females, unmutated VH Ig genes, raised lactate dehydrogenase activity and expression of Zap70 and CD38 [4]. CLL is traditionally treated with a combination of chemotherapeutic reagents, namely, Fludarabine/ Cyclophosphamide/Rituximab (FCR), however treatment remains challenging within the elderly population [1]. Lately the Brutan’s tyrosine kinase inhibitor, Ibrutinib, has shown great promise for the treatment of CLL [5]. However, exceptions are identified as well as treatment resistance prompting further research into CLL treatment strategies [6]

TCFL5 deficiency impairs the pachytene to diplotene transition during spermatogenesis in the mouse

Spermatogenesis is a complex, multistep process during which spermatogonia give rise to spermatozoa. Transcription Factor Like 5 (TCFL5) is a transcription factor that has been described expressed during spermatogenesis. In order to decipher the role of TCFL5 during in vivo spermatogenesis, we generated two mouse models. Ubiquitous removal of TCFL5 generated by breeding TCFL5fl/fl with SOX2-Cre mice resulted in sterile males being unable to produce spermatozoa due to a dramatic alteration of the testis architecture presenting meiosis arrest and lack of spermatids. SYCP3, SYCP1 and H1T expression analysis showed that TCFL5 deficiency causes alterations during pachytene/diplotene transition resulting in a meiotic arrest in a diplotene-like stage. Even more, TCFL5 deficient pachytene showed alterations in the number of MLH1 foci and the condensation of the sexual body. In addition, tamoxifen-inducible TCFL5 knockout mice showed, besides meiosis phenotype, alterations in the spermatids elongation process resulting in aberrant spermatids. Furthermore, TCFL5 deficiency increased spermatogonia maintenance genes (Dalz, Sox2, and Dmrt1) but also increased meiosis genes (Syce1, Stag3, and Morc2a) suggesting that the synaptonemal complex forms well, but cannot separate and meiosis does not proceed. TCFL5 is able to bind to the promoter of Syce1, Stag3, Dmrt1, and Syce1 suggesting a direct control of their expression. In conclusion, TCFL5 plays an essential role in spermatogenesis progression being indispensable for meiosis resolution and spermatids maturatio

Exploring the genetic and genomic connection underlying neurodegeneration with brain iron accumulation and the risk for Parkinson’s disease

Neurodegeneration with brain iron accumulation (NBIA) represents a group of neurodegenerative disorders characterized by abnormal iron accumulation in the brain. In Parkinson’s Disease (PD), iron accumulation is a cardinal feature of degenerating regions in the brain and seems to be a key player in mechanisms that precipitate cell death. The aim of this study was to explore the genetic and genomic connection between NBIA and PD. We screened for known and rare pathogenic mutations in autosomal dominant and recessive genes linked to NBIA in a total of 4481 PD cases and 10,253 controls from the Accelerating Medicines Partnership Parkinsons’ Disease Program and the UKBiobank. We examined whether a genetic burden of NBIA variants contributes to PD risk through single-gene, gene-set, and single-variant association analyses. In addition, we assessed publicly available expression quantitative trait loci (eQTL) data through Summary-based Mendelian Randomization and conducted transcriptomic analyses in blood of 1886 PD cases and 1285 controls. Out of 29 previously reported NBIA screened coding variants, four were associated with PD risk at a nominal p value < 0.05. No enrichment of heterozygous variants in NBIA-related genes risk was identified in PD cases versus controls. Burden analyses did not reveal a cumulative effect of rare NBIA genetic variation on PD risk. Transcriptomic analyses suggested that DCAF17 is differentially expressed in blood from PD cases and controls. Due to low mutation occurrence in the datasets and lack of replication, our analyses suggest that NBIA and PD may be separate molecular entities.National Institutes of Health (NIH

Iron Dyshomeostasis in Neurodegeneration with Brain Iron Accumulation (NBIA): Is It the Cause or the Effect?

: Iron is an essential metal ion implicated in several cellular processes. However, the reactive nature of iron renders this metal ion potentially dangerous for cells, and its levels need to be tightly controlled. Alterations in the intracellular concentration of iron are associated with different neuropathological conditions, including neurodegeneration with brain iron accumulation (NBIA). As the name suggests, NBIA encompasses a class of rare and still poorly investigated neurodegenerative disorders characterized by an abnormal accumulation of iron in the brain. NBIA is mostly a genetic pathology, and to date, 10 genes have been linked to familial forms of NBIA. In the present review, after the description of the principal mechanisms implicated in iron homeostasis, we summarize the research data concerning the pathological mechanisms underlying the genetic forms of NBIA and discuss the potential involvement of iron in such processes. The picture that emerges is that, while iron overload can contribute to the pathogenesis of NBIA, it does not seem to be the causal factor in most forms of the pathology. The onset of these pathologies is rather caused by a combination of processes involving the interplay between lipid metabolism, mitochondrial functions, and autophagic activity, eventually leading to iron dyshomeostasis

Novel variants associated with premature ovarian insufficiency in Russian adolescents

IntroductionWhile variants in hundreds of genes have been linked to premature ovarian insufficiency (POI), monogenic disorders account for fewer than half of idiopathic POI cases in adolescents with 46,XX karyotype. This highlights the need for the further genetic investigation across diverse populations.Patients and methodsWe recruited 63 Russian patients diagnosed with 46,XX POI before age 18. All underwent FMR1 premutation testing and whole-exome sequencing (WES). Copy number variation (CNV) analysis was conducted on WES data. Segregation studies by Sanger sequencing were performed where samples from the patients’ relatives were available.ResultsWe identified variants in 15 genes in 38% of the cohort, including 13 causative genes (FMR1, DCAF17, FOXL2, STAG3, TP63, BNC1, CPEB1, NOBOX, LMNA, FSHR, SPIDR, MCM8, EIF2B2) and 2 candidate genes (MYRF, LATS1). 3.2% of patients carried an FMR1 premutation. WES detected causative single nucleotide variants (SNVs) in 15 patients (17.5% of the cohort). CNV analysis increased the diagnostic yield to 20.6%, identifying 15q25.2 microdeletions (BNC1, CPEB1) in two patients and FSHR exon 2 deletion in one patient with resistant ovary syndrome. Overall, the combination of molecular genetic approaches established a diagnosis of monogenic POI (pathogenic or likely pathogenic variants) in 23.8% of adolescents with normal female karyotype. 5 patients (7.9%) carried variants of unknown significance in FSHR, LMNA, NOBOX, SPIDR, LATS1 genes, warranting further investigation.DiscussionOur findings demonstrate that WES is an effective diagnostic tool for adolescents with POI and should supplement standard karyotyping and FMR1 testing in routine clinical practice. We report several novel variants in POI-associated genes and propose new gene-disease association