Hunter-killer peptides are chimeric synthetic peptides that selectively target specific cell types for an apoptotic death. These peptides, which are models for potential therapeutics, contain a homing sequence for receptor-mediated interactions and a pro-apoptotic sequence. Homing domains have been designed to target angiogenic tumor cells, prostate cells, arthritic tissue and, most recently, adipose tissue. After a receptor-mediated internalization, the apoptotic sequence, which contains D-enantiomer amino acids, initiates apoptosis through mitochondrial membrane disruption. We have begun structure and functional studies on a peptide (HKP1) that specifically targets angiogenic tumor cells for apoptosis. As a model for mitochondrial membrane disruption, we have examined peptide-induced leakage of a calcein fluorophore from large unilamellar vesicles. These experiments demonstrate more potent leakage activity by HKP1 than the peptide lacking the homing domain. Circular dichroism and 2D homonuclear NMR experiments demonstrate that this tumor-specific HKP adopts a left-handed amphipathic helix in association with dodecylphosphorylcholine micelles in a parallel orientation to the lipid-water interface with the homing domain remaining exposed to solvent. The amphipathic helix of the apoptotic domain orients with nonpolar leucine and alanine residues inserting most deeply into the lipid environment.
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US military drone losses in Iran war spur Pentagon call for cheap replacements
Adipose progenitor cells (APCs) are heterogeneous stromal cells and help to maintain metabolic homeostasis. However, the influence of obesity on human APC heterogeneity and the role of APC subpopulations on regulating glucose homeostasis remain unknown. Here, we find that APCs in human visceral adipose tissue contain four subsets. The composition and functionality of APCs are altered in patients with type 2 diabetes (T2D). CD9+CD55low APCs are the subset which is significantly increased in T2D patients. Transplantation of these cells from T2D patients into adipose tissue causes glycemic disturbance. Mechanistically, CD9+CD55low APCs promote T2D development through producing bioactive proteins to form a detrimental niche, leading to upregulation of adipocyte lipolysis. Depletion of pathogenic APCs by inducing intracellular diphtheria toxin A expression or using a hunter-killer peptide improves obesity-related glycemic disturbance. Collectively, our data provide deeper insights in human APC functionality and highlights APCs as a potential therapeutic target to combat T2D. All mice utilized in this study are male.
Brown adipogenesis and thermogenesis in brown and beige adipose tissue (AT) involve vascular remodeling and sympathetic neuronal guidance. Here, we investigated the molecular mechanism coordinating these processes. We used mouse models to identify the molecular target of a peptide CPATAERPC homing to the endothelium of brown and beige AT. We demonstrate that CPATAERPC mimics nerve growth factor (NGF) and identify a low molecular weight isoform of NGF receptor, TrkA, as the CPATAERPC cell surface target. We show that the expression of truncated endothelial TrkA is selective for brown and subcutaneous AT. Analysis of mice with endothelium-specific TrkA knockout revealed the role of TrkA in neuro-vascular coordination supporting the thermogenic function of brown adipocytes. A hunter-killer peptide D-BAT, composed of CPATAERPC and a pro-apoptotic domain, induced cell death in the endothelium and adipocytes. This resulted in thermogenesis impairment, and predisposed mice to obesity and glucose intolerance. We also tested if this treatment can inhibit the tumor recruitment of lipids mobilized from adipocytes from adjacent AT. Indeed, in a mouse model of breast cancer D-BAT suppressed tumor-associated AT lipolysis, which resulted in reduced fatty acid utilization by cancer cells. Our study demonstrates that TrkA signaling in the endothelium supports neuro-vascular coordination enabling beige adipogenesis.
Hunter-killer peptides (HKPs) are synthetic peptides that target specific cell types for apoptosis. These studies report functional and structural characteristics of HKP9, an hunter-killer peptide that specifically targets tumor vasculature with a new apoptotic sequence. Vesicle leakage experiments were performed as a model for membrane perturbing activity. Placement of the homing sequence reduces both cell toxicity and vesicle leakage activity. NMR studies elucidate the conformation and orientation of HKP9 in micelles. The positively charged end of the HKP9 killing sequence is solvent exposed; however, the central portion of the peptide is helical and buried in dodecylphosphorylcholine micelles. The homing sequence is less solvent exposed than in a previously reported tumor-homing peptide. The results suggest that solvent accessibility of the homing sequence should be considered in design of future peptides.
Adipose stromal cells (ASCs) have been identified as a mesenchymal cell population recruited from white adipose tissue (WAT) by tumors and supporting cancer progression. We have previously reported the existence of a non-glycanated decorin isoform (ngDCN) marking mouse ASCs. We identified a peptide CSWKYWFGEC that binds to ngDCN and hence can serve as a vehicle for ASC-directed therapy delivery. We used hunter-killer peptides composed of CSWKYWFGEC and a pro-apoptotic moiety to deplete ASCs and suppress growth of mouse tumors. Here, we report the discovery of the human non-glycanated decorin isoform. We show that CSWKYWFGEC can be used as a probe to identify ASCs in human WAT and tumors. We demonstrate that human ngDCN is expressed on ASC surface. Finally, we validate ngDCN as a molecular target for pharmacological depletion of human ASCs with hunter-killer peptides. We propose that ngDCN-targeting agents could be developed for obesity and cancer treatment.
Progression of many cancers is associated with tumor infiltration by mesenchymal stromal cells (MSC). Adipose stromal cells (ASC) are MSC that serve as adipocyte progenitors and endothelium-supporting cells in white adipose tissue (WAT). Clinical and animal model studies indicate that ASC mobilized from WAT are recruited by tumors. Direct evidence for ASC function in tumor microenvironment has been lacking due to unavailability of approaches to specifically inactivate these cells. Here, we investigate the effects of a proteolysis-resistant targeted hunter-killer peptide D-WAT composed of a cyclic domain CSWKYWFGEC homing to ASC and of a proapoptotic domain KLAKLAK2. Using mouse bone marrow transplantation models, we show that D-WAT treatment specifically depletes tumor stromal and perivascular cells without directly killing malignant cells or tumor-infiltrating leukocytes. In several mouse carcinoma models, targeted ASC cytoablation reduced tumor vascularity and cell proliferation resulting in hemorrhaging, necrosis, and suppressed tumor growth. We also validated a D-WAT derivative with a proapoptotic domain KFAKFAK2 that was found to have an improved cytoablative activity. Our results for the first time demonstrate that ASC, recruited as a component of tumor microenvironment, support cancer progression. We propose that drugs targeting ASC can be developed as a combination therapy complementing conventional cancer treatments.
Hunter-killer peptides combine two activities in a single polypeptide that work in an independent fashion like many other multi-functional, multi-domain proteins. We hypothesize that emergent functions may result from the combination of two or more activities in a single protein domain and that could be a mechanism selected in nature to form moonlighting proteins. We designed moonlighting peptides using the two mechanisms proposed to be involved in the evolution of such molecules (i.e., to mutate non-functional residues and the use of natively unfolded peptides). We observed that our moonlighting peptides exhibited two activities that together rendered a new function that induces cell death in yeast. Thus, we propose that moonlighting in proteins promotes emergent properties providing a further level of complexity in living organisms so far unappreciated.
Fat tissue, overgrowing in obesity, promotes the progression of various carcinomas. Clinical and animal model studies indicate that adipose stromal cells (ASC), the progenitors of adipocytes, are recruited by tumors and promote tumor growth as tumor stromal cells. Here, we investigated the role of ASC in cancer chemoresistance and invasiveness, the attributes of tumor aggressiveness. By using human cell co-culture models, we demonstrate that ASC induce epithelial-mesenchymal transition (EMT) in prostate cancer cells. Our results for the first time demonstrate that ASC interaction renders cancer cells more migratory and resistant to docetaxel, cabazitaxel, and cisplatin chemotherapy. To confirm these findings in vivo, we compared cancer aggressiveness in lean and obese mice grafted with prostate tumors. We show that obesity promotes EMT in cancer cells and tumor invasion into the surrounding fat tissue. A hunter-killer peptide D-CAN, previously developed for targeted ASC ablation, suppressed the obesity-associated EMT and cancer progression. Importantly, cisplatin combined with D-CAN was more effective than cisplatin alone in suppressing growth of mouse prostate cancer allografts and xenografts even in non-obese mice. Our data demonstrate that ASC promote tumor aggressiveness and identify them as a target of combination cancer therapy.
Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.
Duchenne muscular dystrophy (DMD), caused by the loss of dystrophin, remains incurable. Reduction in muscle regeneration with DMD is associated with the accumulation of fibroadipogenic progenitors (FAPs) differentiating into myofibroblasts and leading to a buildup of the collagenous tissue aggravating DMD pathogenesis. Mesenchymal stromal cells (MSCs) expressing platelet-derived growth factor receptors (PDGFRs) are activated in muscle during DMD progression and give rise to FAPs promoting DMD progression. Here, we hypothesized that muscle dysfunction in DMD could be delayed via genetic or pharmacologic depletion of MSC-derived FAPs. In this paper, we test this hypothesis in dystrophin-deficient mdx mice. To reduce fibro/adipose infiltration and potentiate muscle progenitor cells (MPCs), we used a model for inducible genetic ablation of proliferating MSCs via a suicide transgene, viral thymidine kinase (TK), expressed under the Pdgfrb promoter. We also tested if MSCs from fat tissue, the adipose stromal cells (ASCs), contribute to FAPs and could be targeted in DMD. Pharmacological ablation was performed with a hunter-killer peptide D-CAN targeting ASCs. MSC depletion with these approaches resulted in increased endurance, measured based on treadmill running, as well as grip strength, without significantly affecting fibrosis. Although more research is needed, our results suggest that depletion of pathogenic MSCs mitigates muscle damage and delays the loss of muscle function in mouse models of DMD.
The construction and performance of a scroll coil double-resonance probe for solid-state NMR on stationary samples is described. The advantages of the scroll coil at the high resonance frequencies of (1)H and (31)P include: high efficiency, minimal perturbations of tuning by a wide range of samples, minimal RF sample heating of high dielectric samples of biopolymers in aqueous solution, and excellent RF homogeneity. The incorporation of a cable tie cinch for mechanical stability of the scroll coil is described. Experimental results obtained on a Hunter Killer Peptide 1 (HKP1) interacting with phospholipid bilayers of varying lipid composition demonstrate the capabilities of this probe on lossy aqueous samples.
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