Ligand binding‐dependent functions of the lipocalin NLaz: anin vivostudy inDrosophila

Abstract

Lipocalins are small extracellular proteins mostly described as lipid carriers. The Drosophila lipocalin NLaz (neural Lazarillo) modulates the IIS pathway and regulates longevity, stress resistance, and behavior. Here, we test whether a native hydrophobic pocket structure is required for NLaz to perform its functions. We use a point mutation altering the binding pocket (NLaz(L130R)) and control mutations outside NLaz binding pocket. Tryptophan fluorescence titration reveals that NLaz(L130R) loses its ability to bind ergosterol and the pheromone 7(z)-tricosene but retains retinoic acid binding. Using site-directed transgenesis in Drosophila, we test the functionality of the ligand binding-altered lipocalin at the organism level. NLaz-dependent life span reduction, oxidative stress and starvation sensitivity, aging markers accumulation, and deficient courtship are rescued by overexpression of NLaz(WT), but not of NLaz(L130R). Transcriptional responses to aging and oxidative stress show a large set of age-responsive genes dependent on the integrity of NLaz binding pocket. Inhibition of IIS activity and modulation of oxidative stress and infection-responsive genes are binding pocket-dependent processes. Control of energy metabolites on starvation appears to be, however, insensitive to the modification of the NLaz binding pocket.

PROTEINS OF THE INTEGUMENTARY SYSTEM OF THE HONEYBEE, Apis mellifera

“…A type of lipocalin was validated here in the integument of intermediate pharate adults (Pbl phase) at the onset of cuticle pigmentation and may be related to the transport of products for cuticle differentiation. A recent work using a point mutation to alter the ligand binding capacity of a Drosophila lipocalin suggested that this protein binds to ergosterol (the functional equivalent to mammalian cholesterol in insects) and to the pheromone 7(z)‐tricosene, indicating a specific function of lipocalin as a mediator of pheromone signals (Ruiz et al., ). Like the lipocalin here validated, the 7(z)‐tricosene hydrocarbon was identified in the cuticular envelope of A. mellifera pharate adults (Falcón et al., ).…”

Aging without Apolipoprotein D: Molecular and cellular modifications in the hippocampus and cortex

“…Moreover, the expression levels of invertebrate ApoD homologues have been shown to modulate longevity (Hull-Thompson et al, 2009;Ruiz et al, 2011Ruiz et al, , 2012Ruiz et al, , 2014Sanchez et al, 2006).…”

Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress

“…For co-cultures, cells were counted with Countess Automated Cell Counter (Invitrogen). For the exogenous addition of ApoD, human ApoD purified from breast cystic fluid [18] or recombinant human ApoD from E. coli (ProSpec) were added (10 nM) to the cell cultures for 2 h. For long-term treatment (7 days), media was replaced every 48-72 h with freshly added ApoD. The expression constructs used in this work (pcDNA3.1-ApoD; pHSVer-GA;…”

“…These methods were performed as previously described [18,34] The fluorescence spectrum in the presence of ligand was subtracted from DMF baseline obtained mixing the protein with the same amounts of carrier without ligand.…”

“…Some molecules that ApoD can bind are progesterone, arachidonic acid [16], retinol, retinoic acid [17], and sphingomyelin [18]. specifically in the spinal cord, brain and cerebellum [21] but also in the peripheral nervous system (PNS).…”