Biology subjects

Tong, A. H. Y.

Publications and source records attributed to Tong, A. H. Y..

5 recordsLinked to original sources

bioRxiv2022

N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility. In BriefVarland, Silva et al. define that a major cellular role of N-terminal acetylation is shielding proteins from proteasomal degradation by specific ubiquitin ligases. The human N-terminal acetyltransferase NatC protects the neddylation regulator UBE2M from degradation, while overexpression of Drosophila UBE2M/UbcE2M rescues the longevity and motility defects of NatC deletion. HighlightsO_LIN-terminal acetylation by NatC protects proteins from degradation, including UBE2M C_LIO_LIUBR4-KCMF1 targets unacetylated N-terminal Met followed by a hydrophobic residue C_LIO_LIDrosophila NatC is required for adult longevity and motility in elderly C_LIO_LIOverexpression of UBE2M/UbcE2M suppresses Drosophila NatC deletion phenotypes C_LI

molecular biology↗

bioRxiv2021

Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2

SARS-CoV-2, depends on host cell components for replication, therefore the identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection. Such host factors may be necessary for infection and replication of SARS-CoV-2 and, if druggable, presents an attractive strategy for anti-viral therapy. We performed genome wide CRISPR knockout screens in Vero E6 cells and 4 human cell lines including Calu-3, Caco-2, Hek293 and Huh7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while all other host genes identified were cell line specific including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, lipid metabolism, immune pathways and chromatin modulation. Notably, chromatin modulator genes KMT2C and KDM6A in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed. Overall, the network of host factors that have been identified will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate the development of host-directed therapies. IN BRIEFSARS-CoV-2, depends on host cell components for infection and replication. Genome-wide CRISPR screens were performed in multiple human cell lines to elucidate common host dependencies required for SARS-CoV-2 infection. Only ACE2, the cognate SARS-CoV-2 entry receptor, was common amongst cell lines, while all other host genes identified were cell line specific, several of which converged on pathways involved in cell signalling, lipid metabolism, immune pathways, and chromatin modulation. Overall, a network of host factors was identified that will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate productive targeting of host genes and pathways. HIGHLIGHTS- Genome-wide CRISPR screens for SARS-CoV-2 in multiple human cell lines - Identification of wide-ranging cell-type dependent genetic dependencies for SARS-CoV-2 infection - ACE2 is the only common host factor identified across different cell types

systems biology↗

bioRxiv2020

Major functional bias for mitochondrial complexes in genome-wide CRISPR screens

We present FLEX (Functional evaluation of experimental perturbations), a pipeline that leverages several functional annotation resources to establish reference standards for benchmarking human genome-wide CRISPR screen data and methods for analyzing them. We apply FLEX to analyze data from the diverse cell line screens generated by the DepMap project. We identify a dominant mitochondria-associated signal, which our time-resolved CRISPR screens and analysis suggests may reflect screen dynamics and protein stability effects rather than genetic dependencies.

systems biology↗

bioRxiv2019

Systematic mapping of genetic interactions for de novo fatty acid synthesis

The de novo synthesis of fatty acids has emerged as a therapeutic target for various diseases including cancer. While several translational efforts have focused on direct perturbation of de novo fatty acid synthesis, only modest responses have been associated with mono-therapies. Since cancer cells are intrinsically buffered to combat metabolic stress, cells may adapt to loss of de novo fatty acid biosynthesis. To explore cellular response to defects in fatty acid synthesis, we used pooled genome-wide CRISPR screens to systematically map genetic interactions (GIs) in human HAP1 cells carrying a loss-of-function mutation in FASN, which catalyzes the formation of long-chain fatty acids. FASN mutant cells showed a strong dependence on lipid uptake that was reflected by negative GIs with genes involved in the LDL receptor pathway, vesicle trafficking, and protein glycosylation. Further support for these functional relationships was derived from additional GI screens in query cell lines deficient for other genes involved in lipid metabolism, including LDLR, SREBF1, SREBF2, ACACA. Our GI profiles identified a potential role for a previously uncharacterized gene LUR1 (C12orf49) in exogenous lipid uptake regulation. Overall, our data highlights 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.

systems biology↗

bioRxiv2019

Scalable, FACS-Free Genome-Wide Phenotypic Screening

Genome-scale functional genetic screens can identify key regulators of a phenotype of interest, such as determinants of protein expression or modification. Here, we present a rapid, high-throughput approach to phenotypic CRISPR-Cas9 screening. To study factors that modulate the display of CD47 on the cell surface, we processed an entire genome-wide screen containing more than 108 cells in under one hour and maintained high levels of cell viability using a highly scalable cell sorting technology. We robustly identified modulators of CD47 function including QPCTL, an enzyme required for formation of the pyroglutamyl modification at the N-terminus of this protein.

genomics↗