A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID).

The protein translocase of the mitochondrial inner membrane in Trypanosoma brucei, TbTIM17, forms a modular complex in association with several other trypanosome-specific proteins. To identify transiently interacting proximal partner(s) of TbTim17, we used Biotinylation Identification (BioID) by expressing a modified biotin ligase-TbTim17 (BirA∗-TbTim17) fusion protein in T. brucei. BirA∗-TbTim17 was targeted to mitochondria and assembled in the TbTIM complex. In the presence of biotin, BirA∗-TbTim17 biotinylated several mitochondrial proteins. Interestingly, TbHsp84/TbTRAP1, a mitochondrial Hsp90 homolog, was identified as the highest enriched biotinylated proteins. We validated that interaction and colocalization of TbTim17 and TbHsp84 in T. brucei mitochondria by coimmunoprecipitation analysis and confocal microscopy, respectively. TbTim17 association with TbTRAP1 increased several folds during denaturation/renaturation of mitochondrial proteins in vitro, suggesting TbTRAP1 acts as a chaperone for TbTim17 refolding. We demonstrated that knockdown of TbTRAP1 reduced cell growth and decreased the levels of the TbTIM17, TbTim62, and mitochondrial (m)Hsp70 complexes. However, ATPase, VDAC, and Atom69 complexes were minimally affected. Additionally, the steady state levels of TbTim17, TbTim62, and mHsp70 were reduced significantly, but Atom69, ATPase ß, and RBP16 were mostly unaltered due to TbTRAP1 knockdown. Quantitative proteomics analysis also showed significant reduction of TbTim62 along with a few other mitochondrial proteins due to TbTRAP1 knockdown. Finally, TbTRAP1 depletion did not hamper the import of the ectopically expressed TbTim17-2xMyc into mitochondria but reduced its assembly into the TbTIM17 complex, indicating TbTRAP1 is critical for assembly of TbTim17. This is the first report showing the role of TRAP1 in the TIM complex assembly in eukaryotes.

High resolution ultrasound imaging for repeated measure of wound tissue morphometry, biomechanics and hemodynamics under fetal, adult and diabetic conditions.

Non-invasive, repeated interrogation of the same wound is necessary to understand the tissue repair continuum. In this work, we sought to test the significance of non-invasive high-frequency high-resolution ultrasound technology for such interrogation. High-frequency high-resolution ultrasound imaging was employed to investigate wound healing under fetal and adult conditions. Quantitative tissue cellularity and elastic strain was obtained for visualization of unresolved inflammation using Vevo strain software. Hemodynamic properties of the blood flow in the artery supplying the wound-site were studied using color Doppler flow imaging. Non-invasive monitoring of fetal and adult wound healing provided unprecedented biomechanical and functional insight. Fetal wounds showed highly accelerated closure with transient perturbation of wound tissue cellularity. Fetal hemodynamics was unique in that sharp fall in arterial pulse pressure (APP) which was rapidly restored within 48h post-wounding. In adults, APP transiently increased post-wounding before returning to the pre-wounding levels by d10 post-wounding. The pattern of change in the elasticity of wound-edge tissue of diabetics was strikingly different. Severe strain acquired during the early inflammatory phase persisted with a slower recovery of elasticity compared to that of the non-diabetic group. Wound bed of adult diabetic mice (db/db) showed persistent hypercellularity compared to littermate controls (db/+) indicative of prolonged inflammation. Normal skin strain of db/+ and db/db were asynchronous. In db/db, severe strain acquired during the early inflammatory phase persisted with a slower recovery of elasticity compared to that of non-diabetics. This study showcases a versatile clinically relevant imaging platform suitable for real-time analyses of functional wound healing.

Cutaneous Epithelial to Mesenchymal Transition Activator ZEB1 Regulates Wound Angiogenesis and Closure in a Glycemic Status-Dependent Manner.

Epithelial to mesenchymal transition (EMT) and wound vascularization are two critical interrelated processes that enable cutaneous wound healing. Zinc finger E-box binding homeobox 1 (ZEB1), primarily studied in the context of tumor biology, is a potent EMT activator. ZEB1 is also known to contribute to endothelial cell survival as well as stimulate tumor angiogenesis. The role of ZEB1 in cutaneous wounds was assessed using Zeb1+/- mice, as Zeb1-/- mice are not viable. Quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomics was used to elucidate the effect of elevated ZEB1, as noted during hyperglycemia. Under different glycemic conditions, ZEB1 binding to E-cadherin promoter was investigated using chromatin immunoprecipitation. Cutaneous wounding resulted in loss of epithelial marker E-cadherin with concomitant gain of ZEB1. The dominant proteins downregulated after ZEB1 overexpression functionally represented adherens junction pathway. Zeb1+/- mice exhibited compromised wound closure complicated by defective EMT and poor wound angiogenesis. Under hyperglycemic conditions, ZEB1 lost its ability to bind E-cadherin promoter. Keratinocyte E-cadherin, thus upregulated, resisted EMT required for wound healing. Diabetic wound healing was improved in ZEB+/- as well as in db/db mice subjected to ZEB1 knockdown. This work recognizes ZEB1 as a key regulator of cutaneous wound healing that is of particular relevance to diabetic wound complication.