Biology subjects

Dennis, J. W.

Publications and source records attributed to Dennis, J. W..

3 recordsLinked to original sources

bioRxiv2022

SLC3A2 N-glycosylation and alternate evolutionary trajectories for amino acid metabolism

SLC3A2 (4F2hc, CD98) is an adaptor to the SLC7A exchangers and has undergone extensive repositioning of N-glycosylation sites with vertebrate evolution, presumably in synchrony with the species-specific demands of metabolism. The SLC3A2*SLC7A5 heterodimer imports essential amino acids (AA) and thereby stimulates mTOR signaling, while SLC3A2*SLC7A11 imports cystine required for glutathione synthesis and mitigation of oxidative stress. Analysis of SLC3A2 N-glycans revealed stable site-specific profiles of Golgi remodeling, apart from the conserved N365 site where branching and poly-N-acetylglucosamine content were sensitive to the insertion of lost ancestral sites and to metabolism. N-glycans at N381 and N365 stabilized SLC3A2 in the galectin lattice and opposed endocytosis, while N365 which is nearest the membrane, also promoted down-regulation by galectin-driven clathrin-independent endocytosis (glycolipid-lectin GL-Lect). This is the first report of both positive and negative regulation by galectin binding to N-glycans that are strategically positioned in the same membrane glycoproteins. Proteomics analysis in SLC3A2 mutant HeLa cells with induced re-expression of SLC3A2 as bait revealed the canonical non-N-glycosylated interactors, SLC7A5 and SLC7A11 exchangers, but also AA transporters that were dependent on SLC3A2 N-glycosylation, and are themselves, N-glycosylated AA/Na+ symporters (SLC7A1, SLC38A1, SLC38A2, SLC1A4, SLC1A5). The results suggest that the N-glycans on SLC3A2 regulate clustering of SLC7A exchangers with AA/Na+ symporters, thereby promoting Gln/Glu export-driven import of essential AA and cystine, with the potential to adversely impact redox balance. The evolution of modern birds (Neoaves) led to improved control of bioenergetics with the loss of genes including SLC3A2, SLC7A-5, -7, -8, -10, BCAT2, KEAP1, as well as duplications of SLC7A9, SLC7A11 and the Golgi branching enzymes MGAT4B and MGAT4C known to enhance affinities for galectins. Analyzing the fate of these and other genes in the down-sized genomes of birds, spanning [~]10,000 species and >100 Myr of evolution, may reveal the mystery of their longevity with prolonged vitality. Key PointsO_LIGolgi N-glycan remodeling at each site on SLC3A2 differs with the microenvironment. C_LIO_LIThe galectin lattice and GL-Lect mediated endocytosis act as opposing forces on trafficking, controlled by N-glycans at the distal N381 and membrane proximal N365 sites, respectively. C_LIO_LIMutation at N381 or N365 decreased SLC3A2 association with SLC7A5, SLC7A11 and N-glycosylated AA/Na+ symporters as well as the capacity to mitigate stress. C_LIO_LIClustering of SLC3A2*SLC7A exchangers, with AA/Na+ symporter and ATPase Na+/K+ exchanger promotes growth but continuously consumes ATP in non-proliferating cells. C_LIO_LIBird evolution has improved bioenergetics with the deletion of SLC3A2 and associated transporters; - replaced by transporters of keto acids and a re-enforced galectin lattice. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/516651v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1cb7958org.highwire.dtl.DTLVardef@18ba32dorg.highwire.dtl.DTLVardef@861269org.highwire.dtl.DTLVardef@22df0e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell 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↗

bioRxiv2018

The UBR-1 Ubiquitin Ligase Regulates Glutamate Metabolism to Generate Coordinated Motor Pattern in C. elegans

UBR1 is an E3 ubiquitin ligase best known for its ability to target protein degradation by the N-end rule. The physiological functions of UBR family proteins, however, remain not fully understood. We found that the functional loss of C. elegans UBR-1 leads to a specific motor deficit: when adult animals generate reversal movements, A-class motor neurons exhibit synchronized activation, preventing body bending. This motor deficit is rescued by removing GOT-1, a transaminase that converts aspartate to glutamate. Both UBR-1 and GOT-1 are expressed and critically required in premotor interneurons of the reversal motor circuit to regulate the motor pattern. ubr-1 and got-1 mutants exhibit elevated and decreased glutamate level, respectively. These results raise an intriguing possibility that UBR proteins regulate glutamate metabolism, which is critical for neuronal development and signaling.\n\nAuthor SummaryUbiquitin-mediated protein degradation is central to diverse biological processes. The selection of substrates for degradation is carried out by the E3 ubiquitin ligases, which target specific groups of proteins for ubiquitination. The human genome encodes hundreds of E3 ligases; many exhibit sequence conservation across animal species, including one such ligase called UBR1. Patients carrying mutations in UBR1 exhibit severe systemic defects, but the biology behinds UBR1s physiological function remains elusive. Here we found that the C. elegans UBR-1 regulates glutamate level. When UBR-1 is defective, C. elegans exhibits increased glutamate; this leads to synchronization of motor neuron activity, hence defective locomotion when animals reach adulthood. UBR1-mediated glutamate metabolism may contribute to the physiological defects of UBR1 mutations.

neuroscience↗