Project Grant R37AI136697

BIOCHEMISTRY OF SAMHD1-MEDIATED INNATE IMMUNITY RESPONSES - ABSTRACT SAMHD1 IS A HUMAN IMMUNE FACTOR THAT IMPEDES INFECTION OF MYELOID CELLS AND RESTING T LYMPHOCYTES BY HIV- 1. SAMHD1 IS A DEOXYNUCLEOTIDE TRIPHOSPHATE HYDROLASE (DNTPASE), AND THE DISCOVERY OF ITS ANTI-RETROVIRAL ACTIVITY BROUGHT TO LIGHT A NOVEL ANTIVIRAL MECHANISM THAT DEPENDS ON THE ENZYMATICALLY CONTROLLED DEPLETION OF CELLULAR DNTP POOLS. THE ABILITY OF IMMUNE CELLS TO USE DNTP DEPLETION FOR ANTIVIRAL DEFENSE IS PUZZLING, BECAUSE EVEN QUIESCENT AND TERMINALLY DIFFERENTIATED CELLS REQUIRE DEOXYNUCLEOTIDES FOR DNA REPAIR AND MITOCHONDRIAL BIOGENESIS. SAMHD1 IS A KEY REGULATOR OF DNTP CONCENTRATIONS IN NONPROLIFERATING CELLS AND BETTER UNDERSTANDING OF ITS ENZYMATIC PROPERTIES MAY SHED LIGHT ON THE TANTALIZING RELATIONSHIP BETWEEN DNTP METABOLISM AND ANTIVIRAL IMMUNITY. OUR RECENT WORK REVEALED THAT THE 'FACILITATED DNTP DEPLETION' FUNCTION OF SAMHD1 ARISES FROM ITS ALLOSTERIC ACTIVATION MECHANISM, A TWO-STEP PROCESS INVOLVING TETRAMER ASSEMBLY AND SUBSEQUENT ACTIVATION. FACILITATED DNTP DEPLETION ENABLES REGULATORY CROSSTALK BETWEEN BIOSYNTHESIS AND DEPLETION OF DIFFERENT DNTPS AND MANIFESTS AS MORE ROBUST DEPLETION OF SOME NUCLEOTIDES WHEN BIOSYNTHESIS OF CERTAIN OTHERS IS ELEVATED. WE HYPOTHESIZE THAT THE ALLOSTERIC REGULATION OF SAMHD1 IS A KEY COMPONENT TO THE CELLULAR DECISION-MAKING MECHANISM THAT CONTROLS THE SWITCHING BETWEEN MULTIPLE DISCRETE STATES OF CELLULAR DNTP METABOLISM IN NONPROLIFERATING IMMUNE CELLS AND THUS DETERMINES WHETHER AND WHEN THESE CELLS BECOME NON-PERMISSIVE TO HIV INFECTION. IN THIS PROJECT WE SEEK TO ADVANCE OUR UNDERSTANDING OF HOW ALLOSTERIC AND POSTTRANSLATIONAL REGULATION OF SAMHD1 ENABLES DISTINCT STATES OF DNTP METABOLISM BY PURSUING TWO SPECIFIC AIMS. IN AIM 1 WE WILL DELINEATE HOW ENZYMATIC PROPERTIES OF SAMHD1 GIVE RISE TO THE ANTIVIRAL MODALITIES OF DNTP HOMEOSTASIS AND ENABLE IMMUNE AND METABOLIC DECISION-MAKING. WE ALSO SEEK TO ESTABLISH MECHANISTIC RELATIONSHIPS BETWEEN THE DISTINCT STATES OF CELLULAR DNTP METABOLISM AND THE SUSCEPTIBILITY TO HIV INFECTION. IN AIM 2 WE WILL ELUCIDATE THE STRUCTURAL BASIS OF ALLOSTERIC AND POSTTRANSLATIONAL REGULATION OF SAMHD1. OUR RECENT FINDINGS REVEAL THAT SAMHD1 ACTIVATION BY DNTP SUBSTRATES INVOLVES AN INACTIVE TETRAMERIC INTERMEDIATE WITH PARTIAL DNTP OCCUPANCY OF THE ALLOSTERIC SITES. HERE WE WILL FURTHER ELUCIDATE THE CONFORMATIONAL REPERTOIRE OF SAMHD1 ACTIVATION AND HOW IT IS MODULATED BY POSTTRANSLATIONAL MODIFICATIONS, NUCLEIC ACID LIGANDS AND PATHOGENIC MUTATIONS.