👤 Ernst Reichmann

The use of autologous bio-engineered dermo-epidermal skin substitutes (DESS) yields a pivotal opportunity to cover large skin defects in human patients. These skin grafts consist of both epidermal and dermal compartments necessary for robust and permanent functional wound closure. In this study, we investigated the impact of mesenchymal cells derived from different body site origins on the expression pattern of diverse markers within DESS. Human keratinocytes were obtained from interfollicular epidermis, and mesenchymal cells were isolated from foreskin, palmar skin, fat tissue, and tonsils. After expansion, epidermal cells were seeded on collagen I hydrogels containing stromal cells. These human DESS were transplanted on the back of immune-incompetent rats. After 3 weeks, transplants were excised and analyzed using immunohistology techniques. The macroscopic appearance of skin grafts containing tonsil, fat tissue, or palmar derived mesenchymal cells, was similar to substitutes with foreskin derived dermal fibroblasts. All skin grafts had a strong membrane-localized expression of Lingo-1 in the epidermis. Additionally, we observed an intense expression of transglutaminase 5 in upper epidermal cell layers of the skin grafts confirming a proper keratinocyte differentiation. Tropoelastin was localized throughout the dermal compartments and tightly in contact with the dermo-epidermal junction suggesting an advanced maturation of all skin grafts. Our data implicate that stromal cells derived from tonsil, fat tissue, and palmar skin can assume fibroblast functions supporting keratinocyte proliferation and differentiation. These findings indicate that distinct types of mesenchymal cells can be clinically used for skin engineering purposes. Show less

Proteins bind one another in aqua's solution to form tight and specific complexes. Previously we have shown that this is achieved through the modular architecture of the interaction network formed by the interface residues, where tight cooperative interactions are found within modules but not between them. Here we extend this study to cover the entire interface of TEM1 beta-lactamase and its protein inhibitor BLIP using an improved method for deriving interaction maps based on REDUCE to add hydrogen atoms and then by evaluating the interactions using modifications of the programs PROBE, NCI and PARE. An extensive mutagenesis study of the interface residues indeed showed that each module is energetically independent on other modules, and that cooperativity is found only within a module. By solving the X-ray structure of two interface mutations affecting two different modules, we demonstrated that protein-protein binding occur via the structural reorganization of the binding modules, either by a "lock and key" or an induced fit mechanism. To explain the cooperativity within a module, we performed multiple-mutant cycle analysis of cluster 2 resulting in a high-resolution energy map of this module. Mutant studies are usually done in reference to alanine, which can be regarded as a deletion of a side-chain. However, from a biological perspective, there is a major interest to understand non-Ala substitutions, as they are most common. Using X-ray crystallography and multiple-mutant cycle analysis we demonstrated the added complexity in understanding non-Ala mutations. Here, a double mutation replacing the wild-type Glu,Tyr to Tyr,Asn on TEM1 (res id 104,105) caused a major backbone structural rearrangement of BLIP, changing the composition of two modules but not of other modules within the interface. This shows the robustness of the modular approach, yet demonstrates the complexity of in silico protein design. Show less

Protein-protein interactions are essential for life. Yet, our understanding of the general principles governing binding is not complete. In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules. The boundaries between modules are defined by clustering the contact map of the interface. We show that mutations in one module do not affect residues located in a neighboring module. As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small. To the contrary, within their module, mutations cause complex energetic and structural consequences. Experimentally, this phenomenon is shown on the interaction between TEM1-beta-lactamase and beta-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography. Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface. The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. Show less