The insulin peptide B:9-23 is a natural antigen in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). In addition to αβ T cells and B cells, γδ T cells recognize the peptide and infiltrate the pancreatic islets where the peptide is produced within β cells. The peptide contains a cysteine in position 19 (Cys19), which is required for the γδ but not the αβ T cell response, and a tyrosine in position 16 (Tyr16), which is required for both. A peptide-specific mAb, tested along with the T cells, required neither of the two amino acids to bind the B:9-23 peptide. We found that γδ T cells require Cys19 because they recognize the peptide antigen in an oxidized state, in which the Cys19 thiols of two peptide molecules form a disulfide bond, creating a soluble homo-dimer. In contrast, αβ T cells recognize the peptide antigen as a reduced monomer, in complex with the MHCII molecule I-A(g7). Unlike the unstructured monomeric B:9-23 peptide, the γδ-stimulatory homo-dimer adopts a distinct secondary structure in solution, which differs from the secondary structure of the corresponding portion of the native insulin molecule. Tyr16 is required for this adopted structure of the dimerized insulin peptide as well as for the γδ response to it. This observation is consistent with the notion that γδ T cell recognition depends on the secondary structure of the dimerized insulin B:9-23 antigen.

The protein-folding mechanism remains a major puzzle in life science. Purified soluble activation-induced cytidine deaminase (AID) is one of the most difficult proteins to obtain. Starting from inclusion bodies containing a C-terminally truncated version of AID (residues 1-153; AID¹⁵³), an optimized in vitro folding procedure was derived to obtain large amounts of AID¹⁵³, which led to crystals with good quality and to final structural determination. Interestingly, it was found that the final refolding yield of the protein is proline residue-dependent. The difference in the distribution of cis and trans configurations of proline residues in the protein after complete denaturation is a major determining factor of the final yield. A point mutation of one of four proline residues to an asparagine led to a near-doubling of the yield of refolded protein after complete denaturation. It was concluded that the driving force behind protein folding could not overcome the cis-to-trans proline isomerization, or vice versa, during the protein-folding process. Furthermore, it was found that successful refolding of proteins optimally occurs at high pH values, which may mimic protein folding in vivo. It was found that high pH values could induce the polarization of peptide bonds, which may trigger the formation of protein secondary structures through hydrogen bonds. It is proposed that a hydrophobic environment coupled with negative charges is essential for protein folding. Combined with our earlier discoveries on protein-unfolding mechanisms, it is proposed that hydrogen bonds are a primary driving force for de novo protein folding.

The endogenous small GTPase, Rac1, plays a critical role during normal skin wound healing. It remains to be determined whether endogenous Rac1 can be appropriately activated in chronic wounds; if not, whether exogenous Rac1 has therapeutic effects on wound healing. Here we show that Rac1 protein levels were lower in wounds of db/db diabetic mice than wounds in wild type mice during the healing process. To assess the therapeutic potential of exogenous Rac1 in wound healing, we produced a Tat-Rac1 fusion protein that enters into cells through protein transduction. Tat-Rac1 increased proliferation and migration of keratinocytes and dermal fibroblasts in vitro. Topical application of Tat-Rac1 accelerated cutaneous wound closure in vivo in db/db mice as well as wild type mice. Further analyses revealed that Tat-Rac1 had faster re-epithelialization, higher keratinocyte proliferation and migration without an earlier onset of myofibroblast activation than vehicle treated wounds. Tat-Rac1 also reduced inflammation in wounds. Our findings revealed the failure of diabetic wounds to elevate Rac1 expression and suggested a therapeutic strategy utilizing a Rac1-based biologic to compensate for this defect thereby promoting wound healing.

We have reported that JMJD5 and JMJD7 (JMJD5/7) are responsible for the clipping of arginine methylated histone tails to generate "tailless nucleosomes", which could release the pausing RNA polymerase II (Pol II) into productive transcription elongation. JMJD5/7 function as endopeptidases that cleave histone tails specifically adjacent to methylated arginine residues and continue to degrade N-terminal residues of histones via their aminopeptidase activity. Here, we report structural and biochemical studies on JMJD5/7 to understand the basis of substrate recognition and catalysis mechanism by this JmjC subfamily. Recognition between these enzymes and histone substrates is specific, which is reflected by the binding data between enzymes and substrates. High structural similarity between JMJD5 and JMJD7 is reflected by the shared common substrates and high binding affinity. However, JMJD5 does not bind to arginine methylated histone tails with additional lysine acetylation while JMJD7 does not bind to arginine methylated histone tails with additional lysine methylation. Furthermore, the complex structures of JMJD5 and arginine derivatives revealed a Tudor domain-like binding pocket to accommodate the methylated sidechain of arginine, but not lysine. There also exists a glutamine close to the catalytic center, which may suggest a unique imidic acid mediated catalytic mechanism for proteolysis by JMJD5/7.