Kress, J. | bioRxiv Science

bioRxiv2023

Engineering of a biosensor for intracellular aspartate

Aspartate is a limiting metabolite in proliferating cells with its production closely linked to glutamine and mitochondrial metabolism. To date, measuring aspartate concentrations in live cells was deemed impossible. We present iAspSnFR, a genetically-encoded biosensor for intracellular aspartate, engineered by displaying and screening biosensor libraries in HEK293 cells. In live cells, iAspSnFR exhibits a dynamic range of 130% fluorescence change and detects reduced aspartate levels upon glutamine deprivation or glutaminase inhibition. Furthermore, iAspSnFR tracks aspartate uptake by excitatory amino acid transporters, or of asparagine after co-expression of an asparaginase. Importantly, iAspSnFR reports aspartate depletions upon electron transport chain inhibition, and therefore it can serve as a proxy for mitochondrial respiration. Consequently, iAspSnFR can dissect the major cellular pathways of aspartate production, offering immediate applications, particularly in cancer biology, such as screening small molecules targeting aspartate and glutamine metabolism.

cell biology↗

bioRxiv2020

Structural Basis of CD4 Downregulation by HIV-1 Nef

The HIV-1 protein Nef suppresses multiple immune surveillance mechanisms to promote viral pathogenesis1. Individuals infected with HIV-1 encoding defective nef genes do not develop AIDS for decades2,3. A key target of Nef is the cellular receptor CD4. Although essential for viral entry into host cells, CD4 is problematic for the virus later in its replication cycle: CD4 disrupts processing of the viral glycoprotein, Env, inhibiting infectivity4; it interferes with the release of new virions5,6; and it causes vulnerability to superinfection, causing premature cell death and limiting viral productivity7. Furthermore, binding of CD4 to Env exposes otherwise-concealed Env epitopes, rendering infected cells more susceptible to antibody-dependent cellular cytotoxicity and virus particles more susceptible to neutralizing antibodies8-10. HIV-1 has evolved strategies to mitigate these problems. Newly synthesized CD4 is targeted in the endoplasmic reticulum by the viral Vpu protein for proteasomal degradation11. Surface-expressed CD4, in contrast, is targeted by Nef for endocytosis and lysosomal degradation12-15. Nefs effect on CD4 involves hijacking of clathrin adaptor complex 2 (AP2)-dependent endocytosis16,17. Although how Nef associates with a part of the tetrameric AP2 is understood18, a complete understanding of the interaction, especially how CD4 is sequestered by Nef into a complex with AP2, has remained elusive. Here, we present a high-resolution crystal structure that describes the underlying mechanism. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. Strikingly, a pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nefs activities and sensitize the virus to immune clearance

biochemistry↗