Markku Varjosalo

Interaction Proteomics – Master and Commander in Biomedical Research

Markku Varjosalo1

1. University of Helsininki

Proteins are essential macromolecules participating in a wide range of biological processes, including signal transduction, transcription regulation, immune response, and enzymatic catalysis.. Protein-protein interactions (PPIs) are crucial for these activities and over 80% of proteins interact with others to carry out their functions (PMID: 17640003). PPIs play a pivotal role in the organization and function of the proteome, with perturbations often linked to various diseases such as cancer, neurodegeneration, and infectious diseases. PPIs can be stable or transient, with stable interactions forming protein complexes, while transient interactions predominantly occur in signal transduction. Many PPIs are part of larger PPI networks in the cell, and understanding the interactome is critical for comprehending the regulation of biological networks.

Recent advances in mass spectrometry (MS)-based protein interactomics have significantly expanded our understanding of PPIs in cells, with methods that continue to improve in terms of sensitivity, specificity, and resolution. For systematic PPI examination, experimental techniques such as two-hybrid systems, affinity purification, MS, protein chip technology, and computational modeling have been developed. The affinity purification approach uses a tagged protein of interest (POI) as “bait” to bind any interacting proteins (preys), which can be obtained from sources like cell and tissue lysates. The general proteomic workflow using MS is then applied for protein identification. Furthermore, affinity purification can be coupled with crosslinking mass spectrometry and Cryo-EM to obtain a structural proteomics view on protein complexes, providing valuable insights into their architecture and function.

In our research, we have extensively applied interaction proteomics to investigate the dynamic interplay of key cellular signaling molecules, such as protein kinases, phosphatases, and transcription factors, in the context of tens of studies. Our primary focus has been on understanding how these interactions contribute to maintaining functional cells and how their perturbation may lead to various diseases, including cancer and immunological disorders. By utilizing advanced experimental techniques like affinity purification, crosslinking mass spectrometry, and Cryo-EM, we have been able to obtain comprehensive view of protein complexes, shedding light on their architecture and function. This structural proteomics approach has allowed us to gain valuable insights into the molecular mechanisms governing protein interactions and their roles in cellular processes.

One particular focus of our research has been the Commander complex, a 16-protein subunit assembly that plays multiple roles in various intracellular events, including regulation of cell homeostasis, cell cycle, and immune response. The complex is composed of COMMD1-10, CCDC22, CCDC93, DENND10, VPS26C, VPS29, and VPS35L. These proteins are expressed ubiquitously in the human body and have been linked to diseases including Wilson’s disease, atherosclerosis, and several cancers. Despite its importance, the structure and molecular functions of the Commander complex are poorly understood. Through our investigations, we have uncovered the structure and key interactions of the endogenous human Commander complex using cryogenic electron microscopy (cryo-EM) and mass spectrometry-based proteomics. Our results show that the complex is asymmetric, consisting of a stable core of a pseudo-symmetric ring of COMMD proteins 1–10 and a mobile effector consisting of DENND10 and the Retriever sub-complex, constituted by VPS35L, VPS29, and VPS26C. The two halves are scaffolded together by CCDC22 and CCDC93. This study directly confirms the cellular composition of Commander and identifies major interaction interfaces, defining the structure and interaction landscape of the complex.

These findings offer new insights into the known roles, and uncover strong association with cilium, centrosome and centriole functions.

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