Secretome Analysis: Reading Cellular Sign Language to Understand Intercellular Communication

Abstract

•Cell type-specific and proximity biotin labeling enables secretome characterization.•Glycan-mediated secretome analysis requires carefully controlled interpretation.•Recent trends to perform secretome analysis in vivo.•Computational methods are critical to interpret cellular and organismal secretion. A significant portion of mammalian proteomes is secreted to the extracellular space to fulfill crucial roles in cell-to-cell communication. To best recapitulate the intricate and multi-faceted crosstalk between cells in a live organism, there is an ever-increasing need for methods to study protein secretion in model systems that include multiple cell types. In addition, posttranslational modifications further expand the complexity and versatility of cellular communication. This review aims to summarize recent strategies and model systems that employ cellular coculture, chemical biology tools, protein enrichment, and proteomic methods to characterize the composition and function of cellular secretomes. This is all geared towards gaining better understanding of organismal biology in vivo mediated by secretory signaling. A significant portion of mammalian proteomes is secreted to the extracellular space to fulfill crucial roles in cell-to-cell communication. To best recapitulate the intricate and multi-faceted crosstalk between cells in a live organism, there is an ever-increasing need for methods to study protein secretion in model systems that include multiple cell types. In addition, posttranslational modifications further expand the complexity and versatility of cellular communication. This review aims to summarize recent strategies and model systems that employ cellular coculture, chemical biology tools, protein enrichment, and proteomic methods to characterize the composition and function of cellular secretomes. This is all geared towards gaining better understanding of organismal biology in vivo mediated by secretory signaling. The term secretome was first used in the year of 2000 to designate the secretory processes in the bacteria Bacillus subtilis (1Tjalsma H. Bolhuis A. Jongbloed J.D. Bron S. van Dijl J.M. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome.Microbiol. Mol. Biol. Rev. 2000; 64: 515-547Google Scholar) and has since become a generally used term to describe the global group of proteins that are secreted, released, or shed into the extracellular environment by a cell, tissue, organ, or organism at any given time. It has been estimated that the mammalian proteome encompasses nearly 3000 secreted proteins and >2000 that are localized to the plasma membrane, collectively representing some 25% of the proteome (2Uhlen M. Fagerberg L. Hallstrom B.M. Lindskog C. Oksvold P. Mardinoglu A. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 3471260419Google Scholar, 3Uhlen M. Karlsson M.J. Hober A. Svensson A.S. Scheffel J. Kotol D. et al.The human secretome.Sci. Signal. 2019; 12eaaz0274Google Scholar). In addition, trafficking these proteins through the secretory pathway is an energetically expensive process, estimated to involve >250 proteins that are needed after the completion of protein synthesis to translocate secretory proteins through the endoplasmic reticulum (ER) and Golgi, while assembling disulfide bonds and adding N- and O-linked glycans (4Gutierrez J.M. Feizi A. Li S. Kallehauge T.B. Hefzi H. Grav L.M. et al.Genome-scale reconstructions of the mammalian secretory pathway predict metabolic costs and limitations of protein secretion.Nat. Commun. 2020; 11: 68Google Scholar). This expenditure is warranted by the fact that secretory proteins fulfill a range of important extracellular functions that are key to proper functioning of the cell in its niche, organ, and eventually in the entire organism. Specifically, the secretome comprises a large variety of bioactive molecules such as enzymes, hormones, antibodies, and extracellular matrix (ECM) proteins that provide a scaffolding function in the ECM and that play important roles in regulating cell–cell and cell–ECM interactions (3Uhlen M. Karlsson M.J. Hober A. Svensson A.S. Scheffel J. Kotol D. et al.The human secretome.Sci. Signal. 2019; 12eaaz0274Google Scholar, 5Sainio A. Jarvelainen H. Extracellular matrix-cell interactions: focus on therapeutic applications.Cell Signal. 2020; 66109487Google Scholar, 6Naba A. Ten years of extracellular matrix proteomics: accomplishments, challenges, and future perspectives.Mol. Cell Proteomics. 2023; 22100528Google Scholar). In addition, an important class of proteins is constituted by growth factors and cytokines that signal to recipient cells to induce a specific response. This is not only required to maintain tissue homeostasis in healthy tissue (as further stipulated below) but is also crucial in diseases like cancer where secretory proteins constitute the organizing principle among cancer cells, stroma, and immune cells in the tumor microenvironment (7Propper D.J. Balkwill F.R. Harnessing cytokines and chemokines for cancer therapy.Nat. Rev. Clin. Oncol. 2022; 19: 237-253Google Scholar, 8Padgaonkar M. Shendre S. Chatterjee P. Banerjee S. Cancer secretome: finding out hidden messages in extracellular secretions.Clin. Transl. Oncol. 2023; 25: 1145-1155Google Scholar, 9Robinson J.L. Feizi A. Uhlen M. Nielsen J. A systematic investigation of the malignant functions and diagnostic potential of the cancer secretome.Cell Rep. 2019; 26: 2622-26235.e5Google Scholar). Knowing and understanding the proteins in the extracellular space that sustain the mutual interactions among cells therefore not only provides insight in fundamental aspects of tissue maintenance and onset of disease but may also provide clues for intervention by interfering in this network (7Propper D.J. Balkwill F.R. Harnessing cytokines and chemokines for cancer therapy.Nat. Rev. Clin. Oncol. 2022; 19: 237-253Google Scholar, 10Cox T.R. The matrix in cancer.Nat. Rev. Cancer. 2021; 21: 217-238Google Scholar). Furthermore, it is worth noting that proteins in body fluids like blood plasma and cerebrospinal fluid are all secreted by cells and organs throughout the body, putting secretory proteins at center stage for biomarker discovery. The aim of this review is to provide an overview of proteomic strategies that have emerged recently for the enrichment and characterization of secreted proteins, including their posttranslational modifications. Furthermore, we discuss the application of such strategies towards the recapitulation of systemic cellular crosstalk in vivo. Secretory proteins can either function in an autocrine or paracrine fashion to sustain a host of processes that are essential for tissue homeostasis, development, and response to stress. In autocrine signaling, a cell produces a soluble mediator into the extracellular space that binds to a receptor on the same cell, thereby completing a self-sufficient form of auto-regulation. For instance, autocrine production of transforming growth factor-beta (TGFβ) in cancer is a well-studied yet of In the of is by cell and in the to tumor and J. in Scholar). is mediated by soluble factors by cell and on the to that is on cell and for a and network For instance, is of the paracrine in the human body production of is to immune cells, all cell in the body have to and to or in the Rev. 2022; Scholar). This that immune cells can the tumor microenvironment for of in the important and function of cellular secretion is tissue a large of secreted proteins has functions that and it is that cellular can in C. cell and a Transl. 2021; Scholar). the of cells was to that may of therapeutic cell secretome: therapeutic potential by cellular to the of cellular to in body systems where is yet the of secreted factors have been For instance, cell have been in for or such as or and disease S. Cell secretome: and therapeutic for 2020; Scholar). have also been to of proteins on the of in or A. C. C. J. et al.The secretome of a systematic 2020; Scholar). cell have also been in and S. et of cells in and a systematic Transl. 2020; Scholar). these that cellular may a of potential for in only tissue secreted proteins can also tissue and For instance, autocrine is important in biology and and secreted factors such as and are required for in a of therapeutic 2021; Scholar). of such autocrine can in such as and mediated by secreted proteins are also critical in and tissue are in cancer J.M. in can we Cancer Scholar). The is an important organ, in and are to on roles in and in such cells have been to in paracrine signaling, to production of M. paracrine in the of 2022; Scholar). of crosstalk mediated by are in In for a autocrine was to to large bacteria on to have these and that of autocrine and cells and that these are of a of crosstalk H. and of a Cell Biol. 26: Scholar). proteins are not only crucial in tissue homeostasis, but are also key in This where the tumor microenvironment a of cancer cells, stroma, and immune cells that by and factors to play a in the and of D. of 2022; Scholar, The tumor microenvironment cancer 2019; Scholar). ECM by the of and that ECM in can malignant and tumor and and J. A. of extracellular matrix in and Commun. 2020; 11: Scholar). cells are essential of the tumor where that and immune cells play a in tumor L. 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