Publications by authors named "Hala Abdouni"

8 Publications

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A Toolbox for Efficient Proximity-Dependent Biotinylation in Zebrafish Embryos.

Mol Cell Proteomics 2021 Jul 29;20:100128. Epub 2021 Jul 29.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital, Sinai Health, Toronto, Ontario, Canada. Electronic address:

Understanding how proteins are organized in compartments is essential to elucidating their function. While proximity-dependent approaches such as BioID have enabled a massive increase in information about organelles, protein complexes, and other structures in cell culture, to date there have been only a few studies on living vertebrates. Here, we adapted proximity labeling for protein discovery in vivo in the vertebrate model organism, zebrafish. Using lamin A (LMNA) as bait and green fluorescent protein (GFP) as a negative control, we developed, optimized, and benchmarked in vivo TurboID and miniTurbo labeling in early zebrafish embryos. We developed both an mRNA injection protocol and a transgenic system in which transgene expression is controlled by a heat shock promoter. In both cases, biotin is provided directly in the egg water, and we demonstrate that 12 h of labeling are sufficient for biotinylation of prey proteins, which should permit time-resolved analysis of development. After statistical scoring, we found that the proximal partners of LMNA detected in each system were enriched for nuclear envelope and nuclear membrane proteins and included many orthologs of human proteins identified as proximity partners of lamin A in mammalian cell culture. The tools and protocols developed here will allow zebrafish researchers to complement genetic tools with powerful proteomics approaches.
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http://dx.doi.org/10.1016/j.mcpro.2021.100128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8383115PMC
July 2021

A Comprehensive, Flexible Collection of SARS-CoV-2 Coding Regions.

G3 (Bethesda) 2020 09 2;10(9):3399-3402. Epub 2020 Sep 2.

Donnelly Centre, University of Toronto, Toronto, Ontario, Canada

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.
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http://dx.doi.org/10.1534/g3.120.401554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467003PMC
September 2020

Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism.

Nat Metab 2020 06 1;2(6):499-513. Epub 2020 Jun 1.

Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.

The de novo synthesis of fatty acids has emerged as a therapeutic target for various diseases, including cancer. Because cancer cells are intrinsically buffered to combat metabolic stress, it is important to understand how cells may adapt to the loss of de novo fatty acid biosynthesis. Here, we use pooled genome-wide CRISPR screens to systematically map genetic interactions (GIs) in human HAP1 cells carrying a loss-of-function mutation in fatty acid synthase (FASN), whose product catalyses the formation of long-chain fatty acids. FASN-mutant cells show a strong dependence on lipid uptake that is reflected in negative GIs with genes involved in the LDL receptor pathway, vesicle trafficking and protein glycosylation. Further support for these functional relationships is derived from additional GI screens in query cell lines deficient in other genes involved in lipid metabolism, including LDLR, SREBF1, SREBF2 and ACACA. Our GI profiles also identify a potential role for the previously uncharacterized gene C12orf49 (which we call LUR1) in regulation of exogenous lipid uptake through modulation of SREBF2 signalling in response to lipid starvation. Overall, our data highlight the genetic determinants underlying the cellular adaptation associated with loss of de novo fatty acid synthesis and demonstrate the power of systematic GI mapping for uncovering metabolic buffering mechanisms in human cells.
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http://dx.doi.org/10.1038/s42255-020-0211-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566881PMC
June 2020

AID, APOBEC3A and APOBEC3B efficiently deaminate deoxycytidines neighboring DNA damage induced by oxidation or alkylation.

Biochim Biophys Acta Gen Subj 2019 11 9;1863(11):129415. Epub 2019 Aug 9.

Program in Immunology and Infectious Diseases, Department of Biomedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3V6, Canada; Department of Molecular Biology and Biochemistry, Faculty of Science, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada. Electronic address:

Background: AID/APOBEC3 (A3) enzymes instigate genomic mutations that are involved in immunity and cancer. Although they can deaminate any deoxycytidine (dC) to deoxyuridine (dU), each family member has a signature preference determined by nucleotides surrounding the target dC. This WRC (W = A/T, R = A/G) and YC (Y = T/C) hotspot preference is established for AID and A3A/A3B, respectively. Base alkylation and oxidation are two of the most common types of DNA damage induced environmentally or by chemotherapy. Here we examined the activity of AID, A3A and A3B on dCs neighboring such damaged bases.

Methods: Substrates were designed to contain target dCs either in normal WRC/YC hotspots, or in oxidized/alkylated DNA motifs. AID, A3A and A3B were purified and deamination kinetics of each were compared between substrates containing damaged vs. normal motifs.

Results: All three enzymes efficiently deaminated dC when common damaged bases were present in the -2 or -1 positions. Strikingly, some damaged motifs supported comparable or higher catalytic efficiencies by AID, A3A and A3B than the WRC/YC motifs which are their most favored normal sequences. Based on the resolved interactions of AID, A3A and A3B with DNA, we modeled interactions with alkylated or oxidized bases. Corroborating the enzyme assay data, the surface regions that recognize normal bases are predicted to also interact robustly with oxidized and alkylated bases.

Conclusions: AID, A3A and A3B can efficiently recognize and deaminate dC whose neighbouring nucleotides are damaged.

General Significance: Beyond AID/A3s initiating DNA damage, some forms of pre-existing damaged DNA can constitute favored targets of AID/A3s if encountered.
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http://dx.doi.org/10.1016/j.bbagen.2019.129415DOI Listing
November 2019

Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains.

Mol Cell 2019 02 13;73(3):621-638.e17. Epub 2018 Dec 13.

Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. Electronic address:

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.
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http://dx.doi.org/10.1016/j.molcel.2018.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375729PMC
February 2019

A Versatile Lentiviral Delivery Toolkit for Proximity-dependent Biotinylation in Diverse Cell Types.

Mol Cell Proteomics 2018 11 10;17(11):2256-2269. Epub 2018 Jul 10.

From the ‡Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Canada;

Proximity-dependent biotinylation strategies have emerged as powerful tools to characterize the subcellular context of proteins in living cells. The popular BioID approach employs an abortive biotin ligase mutant (R118G; denoted as BirA*), which when fused to a bait protein enables the covalent biotinylation of endogenous proximal polypeptides. This approach has been mainly applied to the study of protein proximity in immortalized mammalian cell lines. To expand the application space of BioID, here we describe a set of lentiviral vectors that enable the inducible expression of BirA*-tagged bait fusion proteins for performing proximity-dependent biotinylation in diverse experimental systems. We benchmark this highly adaptable toolkit across immortalized and primary cell systems, demonstrating the ease, versatility and robustness of the system. We also provide guidelines to perform BioID using these reagents.
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http://dx.doi.org/10.1074/mcp.TIR118.000902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210231PMC
November 2018

DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

Mol Immunol 2018 01 20;93:94-106. Epub 2017 Nov 20.

Program in immunology and Infectious Diseases, Division of Biomedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, A1 B 3V6, Canada. Electronic address:

Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID.
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http://dx.doi.org/10.1016/j.molimm.2017.11.012DOI Listing
January 2018

Zebrafish AID is capable of deaminating methylated deoxycytidines.

Nucleic Acids Res 2013 May 12;41(10):5457-68. Epub 2013 Apr 12.

Program in Immunology and Infectious Diseases, Division of Biomedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St John's, Newfoundland A1B 3V6, Canada.

Activation-induced cytidine deaminase (AID) deaminates deoxycytidine (dC) to deoxyuracil (dU) at immunoglobulin loci in B lymphocytes to mediate secondary antibody diversification. Recently, AID has been proposed to also mediate epigenetic reprogramming by demethylating methylated cytidines (mC) possibly through deamination. AID overexpression in zebrafish embryos was shown to promote genome demethylation through G:T lesions, implicating a deamination-dependent mechanism. We and others have previously shown that mC is a poor substrate for human AID. Here, we examined the ability of bony fish AID to deaminate mC. We report that zebrafish AID was unique among all orthologs in that it efficiently deaminates mC. Analysis of domain-swapped and mutant AID revealed that mC specificity is independent of the overall high-catalytic efficiency of zebrafish AID. Structural modeling with or without bound DNA suggests that efficient deamination of mC by zebrafish AID is likely not due to a larger catalytic pocket allowing for better fit of mC, but rather because of subtle differences in the flexibility of its structure.
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http://dx.doi.org/10.1093/nar/gkt212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664802PMC
May 2013
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