Abstract
Platelet alpha granules, which contain both membrane-bound and secreted proteins, are formed in the megakaryocyte and then delivered along proplatelet elaborations to the newly forming platelets. A critical but poorly understood process is the post-translational processing, sorting and delivery of proteins to alpha granules prior to their delivery to the proplatelet. Defects in the processing and trafficking of platelet proteins can result in reduced platelet formation. In addition, platelet alpha granules may be differentially packaged with pro- or anti-angiogenic proteins, suggesting that protein sorting events that occur before alpha granule formation may significantly impact platelet end-point function. Thus, research at the intersection of protein trafficking and thrombopoiesis may lead to clearer understanding of the mechanisms of proplatelet formation, the mechanisms of disease in inherited platelet disorders, and the mechanisms of platelet function in inflammation, tumor metastasis and angiogenesis. We have used the platelet integrin aIIbb3 as a model protein to investigate the mechanisms of these processes. In this study we identified proteins that interacted with aIIb in either HEK293 cells or stem cell derived megakaryocytes. The purpose of expanding the “interactome” of aIIb is to identify novel protein-protein interactions that are important for protein delivery to the megakaryocyte surface, and thus could be important for alpha granule and proplatelet formation. We used two methods of capturing interacting proteins:
1) a two-cell pull-down assay using Histidine-tagged aIIb and b3 as bait for umbilical cord blood derived megakaryocyte lysate, followed by nickel bead extraction,
2) a crosslinking assay in which photoreactive, crosslinking amino acids are incorporated into growing megakaryocytes, then crosslinked by exposure to UV light. To enrich the population of aIIb residing in the ER and Golgi, a mutant aIIb subunit containing a R858G mutation, which prevents cleavage of pro-aIIb to mature aIIb resulting in intracellular retention, was used as bait in some assays. The captured proteins from both methods were separated by SDS-PAGE and analyzed by mass spectroscopy. Two or more unique peptides were identified for 93 proteins, and 33 proteins were identified in two or more separate experiments. Of these proteins, 45 were potential protein-trafficking proteins, known to interact with aIIb, or of unknown function. Further analysis of one of these proteins, DNAJC10, suggested that it plays a role in aIIbb3 biogenesis and trafficking. DNAJC10 is an HSP40 type protein with a BiP binding domain and a second domain containing two disulfide isomerase motifs. It has been shown to be induced during ER stress, and may assist in delivering misfolded ER proteins to the proteasome for degradation. Immunoprecipitation of aIIb and b3 followed by immunoblot with anti-DNAJC10 mAb revealed protein bands corresponding to the molecular mass of DNAJC10, indicating direct or indirect physical interaction of aIIb and b3 with DNAJC10. siRNA mediated knockdown of DNAJC10 increased aIIbb3 surface expression on human megakaryocytes by 12%. Together these findings indicate that DNAJC10 interacts with aIIbb3 and may play a role in regulating aIIbb3 surface expression. This study offers new insights into the control of aIIbb3 surface expression and further studies may reveal new targets for anti- or pro-integrin therapies.
SARS-CoV-2 Nucleocapsid protein attenuates stress granule formation and alters gene expression via direct interaction with host mRNAs
