ABSTRACT
The ability to precisely control protein complex formation has high utility in the expanding field of biomaterials. Driving protein-protein binding through metal-ligand bridging interactions is a promising method of achieving this goal. Furthermore, the capacity to precisely regulate both complex formation and dissociation enables additional control not available with constitutive protein complexes. Here we describe the design of three metal-controlled protein dimers that are completely monomeric in the absence of metal yet form high-affinity symmetric homodimers in the presence of zinc sulfate. The scaffold used for the designed dimers is the ß1 domain of streptococcal protein G. In addition to forming high-affinity dimers in the presence of metal, the complexes also dissociate upon addition of EDTA. Biophysical characterization revealed that the proteins maintain relatively high thermal stability, bind with high affinity, and are completely monodisperse in the monomeric and dimeric states. High-resolution crystal structures revealed that the dimers adopt the target structure and that the designed metal-binding histidine residues successfully bind zinc and function to drive dimer formation.
Subject(s)
Bacterial Proteins/chemistry , Metals/chemistry , Protein Domains , Protein Multimerization , Bacterial Proteins/metabolism , Binding, Competitive , Circular Dichroism , Crystallography, X-Ray , Drug Design , Metals/metabolism , Models, Molecular , Protein Binding , Zinc Sulfate/chemistry , Zinc Sulfate/metabolismABSTRACT
Foxp3 is the master transcription factor for T regulatory (Treg) cell differentiation and function. This study aimed to test the therapeutic potential of cell penetrating recombinant Foxp3 protein in arthritis. Recombinant Foxp3 protein was fused to a cell penetrating polyarginine (Foxp3-11R) tag to facilitate intracellular transduction. In vitro Foxp3-11R treated CD4(+) T cells showed a 50% increase in suppressive function compared with control protein treated cells. Severity of arthritis in Foxp3-11R treated mice was significantly reduced compared with those treated with a control protein. CD4(+) T cells of lymph nodes and spleen from Foxp3-11R treated mice showed increased levels of Foxp3 expression compared with those of a control protein treated. These results demonstrated that Foxp3-11R can enhance T cell suppressive function and ameliorate experimental arthritis and suggest that cell penetrating recombinant Foxp3 is a potentially useful agent in therapy of arthritis.
Subject(s)
Arthritis, Experimental/therapy , Forkhead Transcription Factors/metabolism , T-Lymphocytes, Regulatory/metabolism , Animals , Arthritis, Experimental/chemically induced , Arthritis, Experimental/metabolism , Cell Membrane/metabolism , Cell Membrane Permeability , Cell Proliferation , Culture Media/metabolism , Female , Forkhead Transcription Factors/administration & dosage , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/therapeutic use , Humans , Interleukin-2 Receptor alpha Subunit/metabolism , Lymph Nodes/metabolism , Lymph Nodes/pathology , Mice , Peptides/metabolism , Protein Transport , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/therapeutic use , Spleen/metabolism , Spleen/pathology , Zymosan/adverse effectsABSTRACT
Streptavidin is widely used as a detection tool in biology research because of its high affinity and specificity binding to biotin. Biotin-streptavidin system has also been explored for detection of infection and tumor in clinical medicine. Here, we show immunosuppressive property of streptavidin on T cell activation and proliferation. Upon CD3 and CD28 stimulation, CD4(+) T cells produce interleukin 2 (IL-2) and express IL-2 receptor α chain (CD25). Addition of streptavidin in T cell culture suppressed IL-2 synthesis and CD25 expression with no cytotoxicity. The immunosuppressive effect of streptavidin was reversed by excessive biotin. Conjugated to a single chain anti-CD7 variable fragment (scFvCD7), streptavidin was directly delivered to T cells and showed substantially more profound suppressive effect on T cell activation. These results suggest that streptavidin could potentially be used as a novel immunomodulator.
