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.

The identification and quantification of misfolded proteins from complex mixtures is important for biological characterization and disease diagnosis, but remains a major bioanalytical challenge. We have developed Hsp40 Affinity Profiling as a bioanalytical approach to profile protein stability in response to cellular stress. In this assay, we ectopically introduce the Hsp40 FlagDNAJB8H31Q into cells and use quantitative proteomics to determine how protein affinity for DNAJB8 changes in the presence of cellular stress, without regard for native clients. Herein, we evaluate potential approaches to improve the performance of this bioanalytical assay. We find that although intracellular crosslinking increases recovery of protein interactors, this is not enough to overcome the relative drop in DNAJB8 recovery. While the J-domain promotes Hsp70 association, it does not affect the yield of protein association with DNAJB8 under basal conditions. By contrast, crosslinking and J-domain ablation both substantially increase relative protein interactor recovery with the structurally distinct Class B Hsp40 DNAJB1 but are completely compensated by poorer yield of DNAJB1 itself. Cellular thermal stress promotes increased affinity between DNAJB8H31Q and interacting proteins, as expected for interactions driven by recognition of misfolded proteins. DNAJB8WT does not demonstrate such a property, suggesting that under stress misfolded proteins are handed off to Hsp70. Hence, we find that DNAJB8H31Q is still our most effective recognition element for the recovery of destabilized client proteins following cellular stress.

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.

Alterations in dopaminergic transmission are associated with neurological disorders, such as depression, autism, and Parkinson's disease. Exposure of rats to ambient fine (FP) or ultrafine (UFP) particles induces oxidative and inflammatory responses in the striatum, a neuronal nucleus with dense dopaminergic innervation and critically involved in the control of motor activity.Objectives: We used an ex vivo system to evaluate the effect of in vivo inhalation exposure to FP and UFP on motor activity and dopaminergic transmission.Materials and Methods: Male adult Wistar rats were exposed to FP, UFP, or filtered air for 8 weeks (subchronic exposure; 5 h/day, 5 days/week) in a particle concentrator. Motor activity was evaluated using the open-field test. Uptake and release of [³H]-dopamine were assessed in striatal synaptosomes, and dopamine D₂ receptor (D₂R) affinity for dopamine was evaluated by the displacement of [³H]-spiperone binding to striatal membranes.Results: Exposure to FP or UFP significantly reduced spontaneous motor activity (ambulatory distance: FP -25%, UFP -32%; ambulatory time: FP -24%, UFP -22%; ambulatory episodes: FP -22%, UFP -30%), decreased [³H]-dopamine uptake (FP -18%, UFP -24%), and increased, although not significantly, [³H]-dopamine release (113.3 ± 16.3 and 138.6 ± 17.3%). Neither FP nor UFP exposure affected D₂R density or affinity for dopamine.Conclusions: These results indicate that exposure to ambient particulate matter reduces locomotion in rats, which could be related to altered striatal dopaminergic transmission: UFP was more potent than FP. Our results contribute to the evidence linking environmental factors to changes in brain function that could turn into neurological and psychiatric disorders.HIGHLIGHTSYoung adult rats were exposed to fine (FP) or ultrafine (UFP) particles for 40 days.Exposure to FP or UFP reduced motor activity.Exposure to FP or UFP reduced dopamine uptake by striatal synaptosomes.Neither D₂R density or affinity for dopamine was affected by FP or UFP.UFP was more potent than FP to exert the effects reported.