RhoGC is a fusion protein from the aquatic fungus , combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from with (GCwCC) and without (GC) the coiled-coil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GC have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2',3'-dideoxyguanosine-5'-triphosphate, MnCl, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzyme concentration. The low affinity observed for GC monomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.
Monosialodihexosylganglioside (GM3)-presenting lipid-coated polymer nanoparticles (NPs) that recapitulate the sequestration of human immunodeficiency virus-1 (HIV-1) particles in CD169+ virus-containing compartments (VCCs) of macrophages were developed as carriers for delivery and sustained release of a combination of two antiretrovirals (ARVs), rilpivirine (RPV) and cabotegravir (CAB). RPV and CAB were co-loaded into GM3-presenting lipid-coated polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) NPs without loss in potency of the drugs. GM3-presenting PLA NPs demonstrated the most favorable release properties and achieved inhibition of HIV-1 infection of primary human macrophages for up to 35 days. Intracellular localization of GM3-presenting PLA NPs in VCCs correlated with retention of intracellular ARV concentrations and sustained inhibition of HIV-1 infection. This work elucidates the design criteria of lipid-coated polymer NPs to utilize CD169+ macrophages as cellular drug depots for eradicating the viral reservoir sites or to achieve long-acting prophylaxis against HIV-1 infection.
Herein, lipid‐coated mesoporous silica nanoparticles (LMSNs) are investigated as biomimetic delivery vehicle for two antiretroviral (ARV) compounds, rilpivirine (RPV), and cabotegravir (CAB). Monosialodihexosylganglioside (GM3) is incorporated into the membrane to facilitate LMSN binding to CD169 (Siglec‐1)‐expressing myeloid cells, that are predominantly expressed in secondary lymphoid tissues in vivo. It is demonstrated that in addition to providing CD169‐binding functionalities, the lipid membrane around the silica core provides stealth properties that dampen the inflammatory cytokine response to ARVs‐loaded LMSN in human monocyte‐derived macrophages. Quantification of RPV and CAB releases from nanoparticles, and assessment of antiviral potency to human immunodeficiency virus 1 (HIV‐1) infection in vitro reveals that RPV and CAB co‐formulated into LMSN retain optimal antiviral potency for 90 days, even upon storage at room temperature, making LMSN an attractive nanoplatform, immune to cold chain requirements. These findings suggest that GM3–LMSN equip the mesoporous silica nanoparticle (MSN) core with lipid‐derived properties for surface passivation and lipid‐mediated binding that are of high interest for achieving an effective delivery of ARVs to tissue reservoirs of HIV‐1 replication.
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