An inhibitory mono-ubiquitylation of the Drosophila initiator caspase Dronc functions in both apoptotic and non-apoptotic pathways

Apoptosis is an evolutionary conserved cell death mechanism, which requires activation of initiator and effector caspases. The Drosophila initiator caspase Dronc, the ortholog of mammalian Caspase-2 and Caspase-9, has an N-terminal CARD domain that recruits Dronc into the apoptosome for activation. In addition to its role in apoptosis, Dronc also has non-apoptotic functions such as compensatory proliferation. One mechanism to control the activation of Dronc is ubiquitylation. However, the mechanistic details of ubiquitylation of Dronc are less clear. For example, monomeric inactive Dronc is subject to non-degradative ubiquitylation in living cells, while ubiquitylation of active apoptosome-bound Dronc triggers its proteolytic degradation in apoptotic cells. Here, we examined the role of non-degradative ubiquitylation of Dronc in living cells in vivo, i.e. in the context of a multi-cellular organism. Our in vivo data suggest that in living cells Dronc is mono-ubiquitylated on Lys78 (K78) in its CARD domain. This ubiquitylation prevents activation of Dronc in the apoptosome and protects cells from apoptosis. Furthermore, K78 ubiquitylation plays an inhibitory role for non-apoptotic functions of Dronc. We provide evidence that not all of the non-apoptotic functions of Dronc require its catalytic activity. In conclusion, we demonstrate a mechanism whereby Dronc's apoptotic and non-apoptotic activities can be kept silenced in a non-degradative manner through a single ubiquitylation event in living cells

Novel Association of the NOTCH Pathway Regulator MIB1 Gene With the Development of Bicuspid Aortic Valve.

IMPORTANCE Nonsyndromic bicuspid aortic valve (nsBAV) is the most common congenital heart valve malformation. BAV has a heritable component, yet only a few causative genes have been identified; understanding BAV genetics is a key point in developing personalized medicine. OBJECTIVE To identify a new gene for nsBAV. DESIGN, SETTING, AND PARTICIPANTS This was a comprehensive, multicenter, genetic association study based on candidate gene prioritization in a familial cohort followed by rare and common association studies in replication cohorts. Further validation was done using in vivo mice models. Study data were analyzed from October 2019 to October 2022. Three cohorts of patients with BAV were included in the study: (1) the discovery cohort was a large cohort of inherited cases from 29 pedigrees of French and Israeli origin; (2) the replication cohort 1 for rare variants included unrelated sporadic cases from various European ancestries; and (3) replication cohort 2 was a second validation cohort for common variants in unrelated sporadic cases from Europe and the US. MAIN OUTCOMES AND MEASURES To identify a candidate gene for nsBAV through analysis of familial cases exome sequencing and gene prioritization tools. Replication cohort 1 was searched for rare and predicted deleterious variants and genetic association. Replication cohort 2 was used to investigate the association of common variants with BAV. RESULTS A total of 938 patients with BAV were included in this study: 69 (7.4%) in the discovery cohort, 417 (44.5%) in replication cohort 1, and 452 (48.2%) in replication cohort 2. A novel human nsBAV gene, MINDBOMB1 homologue MIB1, was identified. MINDBOMB1 homologue (MIB1) is an E3-ubiquitin ligase essential for NOTCH-signal activation during heart development. In approximately 2% of nsBAV index cases from the discovery and replication 1 cohorts, rare MIB1 variants were detected, predicted to be damaging, and were significantly enriched compared with population-based controls (2% cases vs 0.9% controls; P = .03). In replication cohort 2, MIB1 risk haplotypes significantly associated with nsBAV were identified (permutation test, 1000 repeats; P = .02). Two genetically modified mice models carrying Mib1 variants identified in our cohort showed BAV on a NOTCH1-sensitized genetic background. CONCLUSIONS AND RELEVANCE This genetic association study identified the MIB1 gene as associated with nsBAV. This underscores the crucial role of the NOTCH pathway in the pathophysiology of BAV and its potential as a target for future diagnostic and therapeutic intervention.This study was supported in part by grants PID2019-104776RB-I00 and CB16/ 11/00399 (Dr de la Pompa) from the Spanish Ministerio de Ciencia e Innovación (MCIN/ AEI/ 10.13039/501100011033/); a grant from Hadassah France Association (Drs Gilon and Tessler); a grant from the Center for Interdisciplinary Data Science Research of the Hebrew University of Jerusalem (Dr Tessler); grant R35 CA220340 from the National Institutes of Health (Dr Blacklow), and grants R21HL150373, R01HL114823 (Dr Body); BSF grants 2013269 and 2017245 (Drs. Sprinzak and Blacklow); a consolidator grant from the European Research Council (Genomia – ERC-COG-2017-771945; Dr Loeys); the European Reference Network on rare multisystemic vascular disorders (VASCERN - project ID: 769036 partly cofunded by the European Union Third Health Programme (Drs Loeys and Verstraeten); funding from the Outreach project (Dutch Heart Foundation; Dr Luyckx); funding from Heart and Stroke Foundation of Canada/Robert M Freedom Chair of Cardiovascular Science (Dr Mital); sample biobanking and sequencing from Canada were supported by grants from the Leducq Foundation Transatlantic Networks of Excellence grant, and the Ted Rogers Centre for Heart Research; ISF grant 1053/12 (Dr Durst); and grant R01HL150401 from National Heart, Lung, and Blood Institute (Dr Muehlschlegel).S

Examination of K78 mono-ubiquitylation with respect to Dronc’s catalytic activity.

<p>(<b>A</b>) <i>da>Flag-Dronc</i>^<i>wt</i>, <i>da>Flag-Dronc</i>^<i>K78R</i> and <i>da>Flag-Dronc</i>^{<i>K78RC318A</i>} can rescue the lethality of <i>dronc</i>^<i>I29</i> null mutants, whereas <i>da>Flag-Dronc</i>^<i>C318A</i> cannot. (<b>B</b>) Quantification of the number of additional interommatidial cells (IOC) shown in (C-G). Genotypes are indicated. MARCM was used to express transgenic <i>Flag-Dronc</i> constructs in <i>dronc</i>^<i>I29</i> mutant cell clones. n = 10 for <i>dronc</i>^<i>I29</i> MARCM clones, n = 11 for <i>Flag-Dronc</i>^<i>wt</i> in <i>dronc</i>^<i>I29</i> clones, n = 7 for <i>Flag-Dronc</i>^<i>K78R</i> in <i>dronc</i>^<i>I29</i> clones, n = 11 for <i>Flag-Dronc</i>^{<i>K78RC318A</i>} in <i>dronc</i>^<i>I29</i> clones, n = 8 for <i>Flag-Dronc</i>^<i>C318A</i> in <i>dronc</i>^<i>I29</i> clones. Each n corresponds to an average of extra IOC of 3 clones. ns—not significant. (<b>C-G</b>) Pupal retinae 48h after puparium formation expressing the indicated <i>Flag-Dronc</i> constructs in <i>dronc</i>^<i>I29</i> MARCM clones. Clones are marked by GFP and are enclosed by white dashes in the right panels. Examples of extra IOC are marked with yellow arrows. <i>Flag</i>-<i>Dronc</i>^<i>wt</i> and <i>Flag</i>-<i>Dronc</i>^<i>K78R</i> rescue the IOC phenotype of <i>dronc</i> null mutants. However, <i>Flag</i>-<i>Dronc</i>^{<i>K78RC318A</i>} and <i>Flag</i>-<i>Dronc</i>^<i>C318A</i> fail to rescue this phenotype. Quantified in (B). <b>(H)</b> Immunoblotting of lysates of each Flag-Dronc construct in the <i>dronc</i>^<i>I24</i><i>/dronc</i>^<i>I29</i> background shows similar expression levels. For quantifications, the student’s t-test was used. Error bars are SD. * P<0.05; ** P<0.01; *** P<0,001; **** P<0.0001. ns—not significant.</p

Biochemical characterization of Dronc^K78R.

<p>(<b>A</b>) Bacterially expressed 6xHis-Dronc^wt and 6xHis-Dronc^K78R constructs display similar auto-processing activities. 6xHis-Dronc^C318A and 6xHis-Dronc^K78RC318A do not show any auto-processing. (<b>B</b>) <i>In vitro</i> caspase cleavage assays show that 6xHis-Dronc^wt and 6xHis-Dronc^K78R cleave Myc-Drice^C211A with similar activities. 6xHis-Dronc^C318A and 6xHis-Dronc^K78RC318A cannot cleave Myc-Drice^C211A. <b>(C,C’)</b> 3^rd instar lysates of <i>da</i>><i>GFP-Dark</i>+<i>Flag-Dronc</i>^<i>wt</i> and <i>da</i>><i>GFP-Dark</i>+<i>Flag-Dronc</i>^<i>K78R</i> show that in the presence of Dark, Flag-Dronc^K78R is processed significantly more than Flag-Dronc^wt. In (C’), the average of 4 immunoblots is plotted. (<b>D</b>) GFP-Dark interacts with Flag-Dronc^wt and Flag-Dronc^K78R. GFP-immunoprecipitates of 3^rd instar larval extracts from <i>da</i>><i>GFP-Dark</i>+<i>Flag-Dronc</i>^<i>wt</i>, <i>da</i>><i>GFP-Dark</i>+<i>Flag-Dronc</i>^<i>K78R</i> and <i>da</i>><i>GFP-Dark</i>+<i>EV</i> (<i>Flag-Empty Vector</i>) animals, probed with anti-GFP antibody (upper panel) and anti-Flag antibody (lower panel). There is a stronger interaction between GFP-Dark and Flag-Dronc^K78R, resulting in significantly more efficient procession of Flag-Dronc^K78R compared to Flag-Dronc^wt. (<b>D’</b>) Relative ratio of processed and unprocessed Flag-Dronc proteins in the Dark apoptosome. Flag-Dronc^K78R is more efficiently processed than Flag-Dronc^wt. The average of 3 immunoblots is plotted. For quantifications, the student’s t-test was used. Error bars are SD. * P<0.05.</p