Macrophages play a vital role in the innate immune system, identifying and destroying unwanted cells. However, it has been difficult to attain a comprehensive understanding of macrophage protein abundance due to technical limitations. In addition, it remains unclear how changes in proteome composition are linked to phagocytic activity. In this study we developed methods to derive human macrophages and prepare them for mass spectrometry analysis in order to more-deeply understand the proteomic consequences of macrophage stimulation. Interferon gamma (IF-g), an immune stimulating cytokine, was used to induce macrophage activation, increasing phagocytosis of cancer cells by 2-fold. These conditions were used to perform comparative shotgun proteomics between resting macrophages and stimulated macrophages with increased phagocytic activity. Our analysis revealed that macrophages bias their protein production toward biological processes associated with phagocytosis and antigen processing in response to stimulation. We confirmed our findings by antibody-based western blotting experiments, validating both previously reported and novel proteins of interest. In addition to whole protein changes, we evaluated active protein synthesis by treating cells with the methionine surrogate probe homopropargylglycine (HPG). We saw increased rates of HPG incorporation during phagocytosis-inducing stimulation, suggesting protein synthesis rates are altered by stimulation. Together our findings provide the most comprehensive proteomic insight to date into primary human macrophages. We anticipate that this data can be used as a launchpoint to generate new hypotheses about innate immune function.

Voltage-gated ion channels (VGICs) comprise multiple structural units, the assembly of which is required for function^1,2. Structural understanding of how VGIC subunits assemble and whether chaperone proteins are required is lacking. High-voltage-activated calcium channels (Ca_Vs)^3,4 are paradigmatic multisubunit VGICs whose function and trafficking are powerfully shaped by interactions between pore-forming Ca_V1 or Ca_V2 Ca_Vα₁ (ref. ³), and the auxiliary Ca_Vβ⁵ and Ca_Vα₂δ subunits^6,7. Here we present cryo-electron microscopy structures of human brain and cardiac Ca_V1.2 bound with Ca_Vβ₃ to a chaperone-the endoplasmic reticulum membrane protein complex (EMC)^8,9-and of the assembled Ca_V1.2-Ca_Vβ₃-Ca_Vα₂δ-1 channel. These structures provide a view of an EMC-client complex and define EMC sites-the transmembrane (TM) and cytoplasmic (Cyto) docks; interaction between these sites and the client channel causes partial extraction of a pore subunit and splays open the Ca_Vα₂δ-interaction site. The structures identify the Ca_Vα₂δ-binding site for gabapentinoid anti-pain and anti-anxiety drugs⁶, show that EMC and Ca_Vα₂δ interactions with the channel are mutually exclusive, and indicate that EMC-to-Ca_Vα₂δ hand-off involves a divalent ion-dependent step and Ca_V1.2 element ordering. Disruption of the EMC-Ca_V complex compromises Ca_V function, suggesting that the EMC functions as a channel holdase that facilitates channel assembly. Together, the structures reveal a Ca_V assembly intermediate and EMC client-binding sites that could have wide-ranging implications for the biogenesis of VGICs and other membrane proteins.