Dissecting the calcium-induced differentiation of human primary keratinocytes stem cells by integrative and structural network analyses

The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55%) are composed of non-dynamic and dynamic gene products (‘di-chromatic’), 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation.
Author Summary A key challenge in cellular network biology is to understand how protein complexes are cell-type or condition-specific assembled and disassembled. Cell differentiation is a major cellular reorganization bringing about fundamental changes in the new differentiated cell type. As many genes are expressed throughout all stages and only their expression levels differ, the question arises of how specific functions can be mediated. Here, focusing on the calcium-induced differentiation of primary human keratinocytes, we describe motifs of protein complex assemblies. We found that a large proportion of complexes contain both proteins expressed at similar levels in all stages of differentiation (non-dynamically expressed) and proteins with variable expression between (dynamically expressed). Using structural information we found that subunits tend to be replaced at structural overlapping surfaces of proteins. When applying our concepts to a manually annotated large TNF/NFkB signaling complex, we find a stable core associated with both a dynamically changing module and several stable modules. We propose this as a ‘constant signalosome ready to work,’ where a stable core is associated with a dynamic periphery. Altogether, our analysis highlights the importance of understanding the dynamic assembly and disassembly of complexes, taking 3D structural information into consideration, rather than only considering networks of individual proteins.