Showing papers by "Gilles A. Lajoie published in 2019"

The mammalian CTLH complex is an E3 ubiquitin ligase that targets its subunit muskelin for degradation.

Abstract: The multi-subunit C-terminal to LisH (CTLH) complex is the mammalian homologue of the yeast Gid E3 ubiquitin ligase complex. In this study, we investigated the human CTLH complex and characterized its E3 ligase activity. We confirm that the complex immunoprecipitated from human cells comprises RanBPM, ARMC8 α/β, muskelin, WDR26, GID4 and the RING domain proteins RMND5A and MAEA. We find that loss of expression of individual subunits compromises the stability of other complex members and that MAEA and RMND5A protein levels are interdependent. Using in vitro ubiquitination assays, we demonstrate that the CTLH complex has E3 ligase activity which is dependent on RMND5A and MAEA. We report that the complex can pair with UBE2D1, UBE2D2 and UBE2D3 E2 enzymes and that recombinant RMND5A mediates K48 and K63 poly-ubiquitin chains. Finally, we show a proteasome-dependent increase in the protein levels of CTLH complex member muskelin in RMND5A KO cells. Furthermore, muskelin ubiquitination is dependent on RMND5A, suggesting that it may be a target of the complex. Overall, we further the characterization of the CTLH complex as an E3 ubiquitin ligase complex in human cells and reveal a potential autoregulation mechanism.

Proteomics-Derived Biomarker Panel Improves Diagnostic Precision to Classify Endometrioid and High-grade Serous Ovarian Carcinoma

Abstract: Purpose: Ovarian carcinomas are a group of distinct diseases classified by histotypes. As histotype-specific treatment evolves, accurate classification will become critical for optimal precision medicine approaches. Experimental Design: To uncover differences between the two most common histotypes, high-grade serous (HGSC) and endometrioid carcinoma, we performed label-free quantitative proteomics on freshly frozen tumor tissues (HGSC, n = 10; endometrioid carcinoma, n = 10). Eight candidate protein biomarkers specific to endometrioid carcinoma were validated by IHC using tissue microarrays representing 361 cases of either endometrioid carcinoma or HGSC. Results: More than 500 proteins were differentially expressed (P Conclusions: In summary, these findings support the use of multi-marker panels for the differential diagnosis of difficult cases resembling endometrioid carcinoma and HGSC.

Untargeted metabolomics reveals N, N, N-trimethyl-L-alanyl-L-proline betaine (TMAP) as a novel biomarker of kidney function

Abstract: The diagnosis and prognosis of chronic kidney disease (CKD) currently relies on very few circulating small molecules, which can vary by factors unrelated to kidney function. In end-stage renal disease (ESRD), these same small molecules are used to determine dialysis dose and dialytic clearance. Therefore, we aimed to identify novel plasma biomarkers to estimate kidney function in CKD and dialytic clearance in ESRD. Untargeted metabolomics was performed on plasma samples from patients with a single kidney, non-dialysis CKD, ESRD and healthy controls. For ESRD patients, pre- and post-dialysis plasma samples were obtained from several dialysis modalities. Metabolomics analysis revealed over 400 significantly different features in non-dialysis CKD and ESRD plasma compared to controls while less than 35 features were significantly altered in patients with a single kidney. N,N,N-trimethyl-L-alanyl-L-proline betaine (TMAP, AUROC = 0.815) and pyrocatechol sulfate (AUROC = 0.888) outperformed creatinine (AUROC = 0.745) in accurately identifying patients with a single kidney. Several metabolites accurately predicted ESRD; however, when comparing pre-and post-hemodialysis, TMAP was the most robust biomarker of dialytic clearance for all modalities (AUROC = 0.993). This study describes TMAP as a novel potential biomarker of kidney function and dialytic clearance across several hemodialysis modalities.

Loss of ENT1 increases cell proliferation in the annulus fibrosus of the intervertebral disc

Abstract: Mice lacking equilibrative nucleoside transporter 1 (ENT1 -/- ) demonstrate progressive calcification of spinal tissues including the annulus fibrosus (AF) of the intervertebral disc (IVD). We previously established ENT1 as the primary nucleoside transporter in the AF and demonstrated dysregulation of biomineralization pathways. To identify cellular pathways altered by loss of ENT1, we conducted microarray analysis of AF tissue from wild-type (WT) and ENT1 -/- mice before calcification (2 months of age) and associated with calcification (6 months of age). Bioinformatic analyses identified cell cycle dysregulation in ENT1 -/- AF tissues and implicated the E2f family of transcription factors as potential effectors. Quantitative polymerase chain reaction analysis confirmed increased expression of multiple E2f transcription factors and E2f interacting proteins ( Rb1 and Cdk2) in ENT1 -/- AF cells compared with WT at 6 months of age. At this time point, ENT1 -/- AF tissues showed increased JNK MAPK pathway activation, CDK1, minichromosome maintenance complex component 5 (Mcm5), and proliferating cell nuclear antigen (PCNA) protein expression, and PCNA-positive proliferating cells compared with WT controls. The current study demonstrates that loss of ENT1-mediated adenosine transport leads to increased cell proliferation in the AF of the IVD.

Human Multipotent Stromal Cell Secreted Effectors Accelerate Islet Regeneration.

Abstract: Human multipotent stromal cells (hMSC) can induce islet regeneration after transplantation via the secretion of proteins that establish an islet regenerative niche. However, the identity of hMSC-secreted signals and the mechanisms by which pancreatic islet regeneration is induced remain unknown. Recently, mammalian pancreatic α-cells have been shown to possess considerable plasticity, and differentiate into β-like cells after near complete β-cell loss or overexpression of key transcriptional regulators. These studies have generated new excitement that islet regeneration during diabetes may be possible if we can identify clinically applicable stimuli to modulate these key regulatory pathways. Herein, we demonstrate that intrapancreatic-injection of concentrated hMSC-conditioned media (CM) stimulated islet regeneration without requiring cell transfer. hMSC CM-injection significantly reduced hyperglycemia, increased circulating serum insulin concentration, and improved glucose tolerance in streptozotocin-treated mice. The rate and extent of endogenous β-cell mass recovery was dependent on total protein dose administered and was further augmented by the activation of Wnt-signaling using GSK3-inhibition during CM generation. Intrapancreatic hMSC CM-injection immediately set in motion a cascade of regenerative events that included the emergence of proliferating insulin+ clusters adjacent to ducts, NKX6.1 expression in glucagon+ cells at days 1-4 suggesting the acquisition of β-cell phenotype by α-cells, and accelerated β-cell maturation with increased MAFA-expression for >1 month postinjection. Discovery and validation of islet regenerative hMSC-secreted protein may lead to the development of cell-free regenerative therapies able to tip the balance in favor of β-cell regeneration versus destruction during diabetes. Stem Cells 2019;37:516-528.