ABSTRACT
The activity-regulated cytoskeleton-associated protein (Arc) is a complex regulator of synaptic plasticity in glutamatergic neurons. Understanding its molecular function is key to elucidate the neurobiology of memory and learning, stress regulation, and multiple neurological and psychiatric diseases. The recent development of anti-Arc nanobodies has promoted the characterization of the molecular structure and function of Arc. This study aimed to validate two anti-Arc nanobodies, E5 and H11, as selective modulators of the human Arc N-lobe (Arc-NL), a domain that mediates several molecular functions of Arc through its peptide ligand binding site. The structural characteristics of recombinant Arc-NL-nanobody complexes were solved at atomic resolution using X-ray crystallography. Both anti-Arc nanobodies bind specifically to the multi-peptide binding site of Arc-NL. Isothermal titration calorimetry showed that the Arc-NL-nanobody interactions occur at nanomolar affinity, and that the nanobodies can displace a TARPγ2-derived peptide from the binding site. Thus, both anti-Arc-NL nanobodies could be used as competitive inhibitors of endogenous Arc ligands. Differences in the CDR3 loops between the two nanobodies indicate that the spectrum of short linear motifs recognized by the Arc-NL should be expanded. We provide a robust biochemical background to support the use of anti-Arc nanobodies in attempts to target Arc-dependent synaptic plasticity. Function-blocking anti-Arc nanobodies could eventually help unravel the complex neurobiology of synaptic plasticity and allow to develop diagnostic and treatment tools.
Subject(s)
Cytoskeletal Proteins , Nerve Tissue Proteins , Single-Domain Antibodies , Humans , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/immunology , Single-Domain Antibodies/metabolism , Binding Sites , Cytoskeletal Proteins/metabolism , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/immunology , Ligands , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/immunology , Crystallography, X-Ray , Protein Binding , Models, Molecular , Amino Acid SequenceABSTRACT
The use of a proper sample processing methodology for maximum proteome coverage and high-quality quantitative data is an important choice to make before initiating a liquid chromatography-mass spectrometry (LC-MS)-based proteomics study. Popular sample processing workflows for proteomics involve in-solution proteome digestion and single-pot, solid-phase-enhanced sample preparation (SP3). We tested them on both HeLa cells and human plasma samples, using lysis buffers containing SDS, or guanidinium hydrochloride. We also studied the effect of using commercially available depletion mini spin columns before SP3, to increase proteome coverage in human plasma samples. Our results show that the SP3 protocol, using either buffer, achieves the highest number of quantified proteins in both the HeLa cells and plasma samples. Moreover, the use of depletion mini spin columns before SP3 results in a two-fold increase of quantified plasma proteins. With additional fractionation, we quantified nearly 1400 proteins, and examined lower-abundance proteins involved in neurodegenerative pathways and mitochondrial metabolism. Therefore, we recommend the use of the SP3 methodology for biological sample processing, including those after depletion of high-abundance plasma proteins.
