Drug binding to the P2Y1 receptor prevents phosphorylation and competes with an endogenous ligand.

The Parkinson's disease-associated gene, LRRK2, is also associated with immune disorders and infectious disease and is expressed in immune subsets. Here, we characterize a platform for interrogating the expression and function of endogenous LRRK2 in authentic human phagocytes using human induced pluripotent stem cell-derived macrophages and microglia. Endogenous LRRK2 is expressed and upregulated by interferon-g in these cells, including a 187-kDa cleavage product. Using LRRK2 knockout and G2019S isogenic repair lines, we find that LRRK2 is not involved in initial phagocytic uptake of bioparticles but is recruited to LAMP1 + /RAB9 + ''maturing'' phagosomes, and LRRK2 kinase inhibition enhances its residency at the phagosome. Importantly, LRRK2 is required for RAB8a and RAB10 recruitment to phagosomes, implying that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in these professional phagocytes.

CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA), a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the–LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies.

Olefin cross-metathesis (CM) is a viable reaction for the modification of alkene-containing proteins. Although allyl sulfide or selenide side-chain motifs in proteins can critically enhance the rate of CM reactions, no efficient method for their site-selective genetic incorporation into proteins has yet been reported. Here, through the systematic evaluation of olefin-bearing unnatural amino acids for their metabolic incorporation, we have discovered S-allylhomocysteine (Ahc) as a genetically encode-able Met analogue that is both processed by translational cellular machinery and is also a privileged CM substrate residue in proteins. In this way, Ahc was used for efficient Met-codon reassignment in a Met-auxotrophic strain of E. coli (B834 (DE3)), as well as metabolic labeling of protein in human cells and was reactive towards CM in several representative proteins. This expands the use of CM in the tool kit for 'tagand-modify' functionalization of proteins. Alkenes can be installed into proteins by incorporation of some unnatural amino acids. 1-4 However, although aliphatic alkene-containing amino acids such as homoallylglycine (Hag) 4 are reactive in both self-metathesis and crossmetathesis reactions as monomeric, protected amino acids in organic solvents 5 and can be metabolically incorporated into proteins, they are unreactive in CM reactions in aqueous media. 6 On the other hand, chemically-installed 7 unnatural amino acids such as S-allylcysteine (Sac) 6 ASSOCIATED CONTENT Supporting Information Full procedures and protein ESI-MS. Material is available free of charge via the Internet at http://pubs.acs.org.

SummaryThe Parkinson’s disease-associated gene, LRRK2, is also associated with immune disorders and infectious disease, and is expressed in immune subsets. Here, we characterise a platform for interrogating the expression and function of endogenous LRRK2 in authentic human phagocytes, using human induced Pluripotent Stem Cell-derived macrophages and microglia. Endogenous LRRK2 is expressed and upregulated by interferon-γ in these cells, including a 187kD cleavage product. Using LRRK2 knockout and G2019S isogenic repair lines, we find that LRRK2 is not involved in initial phagocytic uptake of bioparticles, but is recruited to LAMP1(+)/Rab9(+) ‘maturing’ phagosomes, and LRRK2 kinase inhibition enhances its residency at the phagosome. Importantly, LRRK2 is required for Rab8a and Rab10 recruitment to phagosomes, implying that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in these professional phagocytes.