Hsp72 (HSPA1A) Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment

Type 2 diabetes is a growing public health concern and accounts for approximately 90% of all the cases of diabetes. Besides insulin resistance, type 2 diabetes is characterized by a deficit in β-cell mass as a result of misfolded human islet amyloid polypeptide (h-IAPP) which forms toxic aggregates that destroy pancreatic β-cells. Heat shock proteins (HSP) play an important role in combating the unwanted self-association of unfolded proteins. We hypothesized that Hsp72 (HSPA1A) prevents h-IAPP aggregation and toxicity. In this study, we demonstrated that thermal stress significantly up-regulates the intracellular expression of Hsp72, and prevents h-IAPP toxicity against pancreatic β-cells. Moreover, Hsp72 (HSPA1A) overexpression in pancreatic β-cells ameliorates h-IAPP toxicity. To test the hypothesis that Hsp72 (HSPA1A) prevents aggregation and fibril formation, we established a novel C. elegans model that expresses the highly amyloidogenic human pro-IAPP (h-proIAPP) that is implicated in amyloid formation and β-cell toxicity. We demonstrated that h-proIAPP expression in body-wall muscles, pharynx and neurons adversely affects C. elegans development. In addition, we demonstrated that h-proIAPP forms insoluble aggregates and that the co-expression of h-Hsp72 in our h-proIAPP C. elegans model, increases h-proIAPP solubility. Furthermore, treatment of transgenic h-proIAPP C. elegans with ADAPT-232, known to induce the expression and release of Hsp72 (HSPA1A), significantly improved the growth retardation phenotype of transgenic worms. Taken together, this study identifies Hsp72 (HSPA1A) as a potential treatment to prevent β-cell mass decline in type 2 diabetic patients and establishes for the first time a novel in vivo model that can be used to select compounds that attenuate h-proIAPP aggregation and toxicity

A mouse model for triple-negative breast cancer tumor-initiating cells (TNBC-TICs) exhibits similar aggressive phenotype to the human disease

<p>Abstract</p> <p>Background</p> <p>Triple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems.</p> <p>Methods</p> <p>To understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis.</p> <p>Results</p> <p>We isolated tumor-initiating cells (TICs) by sorting for CD24⁺/CD44^high/ALDH1⁺cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24⁺/CD44^high/ALDH1⁺cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24^-/CD44^-/ALDH1^-cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A).</p> <p>Conclusions</p> <p>Taken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC.</p

Heat Shock Protein 90 in Human Diseases and Disorders [electronic resource] /

The book Heat Shock Protein 90 in Human Diseases and Disorders provides the most comprehensive review on contemporary knowledge on the role of HSP90. Using an integrative approach, the contributors provide a synopsis of novel mechanisms, previously unknown signal transduction pathways. To enhance the ease of reading and comprehension, this book has been subdivided into various section including; Section I, reviews current progress on our understanding Oncogenic Aspects of HSP90; Section II, focuses on Bimolecular Aspects of HSP90; Section III, emphasizes and HSP90 in Natural Products Development and Section IV; give the most up to date reviews on Clinical Aspects of HSP90. Key basic and clinical research laboratories from major universities, academic medical hospitals, biotechnology and pharmaceutical laboratories around the world have contributed chapters that review present research activity and importantly project the field into the future. The book is a must read for graduate students. medical students, basic science researchers and postdoctoral scholars in the fields of Translational Medicine, Clinical Research, Human Physiology, Biotechnology, Natural Products, Cell & Molecular Medicine, Pharmaceutical Scientists and Researchers involved in Drug Discovery.SECTION I: ONCOGENIC ASPECTS OF HSP90 -- Chapter 1. Regulatory Roles of HSP90-rich Extracellular Vesicles -- Chapter 2. HSP90-Based Heterocomplex as Essential Regulator for Cancer Disease -- Chapter 3. Therapeutic Potential of Heat Shock Protein 90 Inhibitors in Colorectal Cancer -- Chapter 4. Hsp90 in the Migration of Primordial Germ Cells: A Model to Study Long-Distance Cell Migration and Perhaps Cancer? -- Chapter 5. Role of Heat Shock Protein 90 in Mammary Tumorigenesis -- Chapter 6. Role of HSP90 Inhibitors in the Treatment of Cancer -- Chapter 7. p53-HSP90 Axis in Human Cancer -- Chapter 8. HSP90 and its Inhibitors for Cancer Therapy: Use of Nano-delivery System to Improve its Clinical Application -- Chapter 9. Hsp90 is a Pivotal Player in Retinal Disease and Cancer -- Chapter 10. Targeting Hsp-90 Related Disease Entities for Therapeutic Development -- Chapter 11. HSP90: A Key Player in Metal-Induced Carcinogenesis? -- Section II: Biomolecular Aspects of HSP90 -- Chapter 12. Hsp90 and its Role in Heme-Maturation of Client Proteins: Implications for Human Diseases -- Chapter 13. Moonlighting Functions of Heat Shock Protein 90 -- Chapter 14. Hsp90 as a Member of Dicarboxylate Clamp TPR Protein Interaction Network: Implication in Human Diseases and Prospect as a Drug Target -- Chapter 15. ‘Complex World’ of Hsp90 Co-chaperone R2TP -- Chapter 16. Functions of SGT1, a Co-chaperone -- Chapter 17. Sti1/Hop Plays a Pivotal Role in Hsp90 Regulation beyond Bridging Hsp70 -- Section III: HSP90 in Natural Products Development.-Chapter 18. Hsp90: A Target for Susceptibilities and Substitutions in Biotechnological and Medicinal Application -- Chapter 19. Screening Technique for Heat Shock Protein 90 Inhibitors from Natural Products -- Chapter 20. Therapeutic Effects and Related Molecular Mechanisms of Celastrol, a Triterpenoid Natural Compound and Novel HSP90 Inhibitor Extracted from Plants of the Celastraceae Family -- Section IV: Clinical Aspects of HSP90 -- Chapter 21. Hsp90 Chaperone in Disease -- Chapter 22. Theranostic Implications of Heat Shock Proteins in Idiopathic Pulmonary Fibrosis -- Chapter 23. Heat Shock Protein 90 and Reproduction in Female Animals: Ovary, Oocyte and Early Embryo -- Chapter 24. Heat Shock Protein 90 in Severe Trauma -- Chapter 25. HSP90 - Is there an Unknown Role in Pain Neurobiology -- Chapter 26. Heat Shock Protein 90 in Kidney Stone Disease -- Chapter 27. HSP90 et al. - Chaperome and Proteostasis Deregulation in Human Disease.The book Heat Shock Protein 90 in Human Diseases and Disorders provides the most comprehensive review on contemporary knowledge on the role of HSP90. Using an integrative approach, the contributors provide a synopsis of novel mechanisms, previously unknown signal transduction pathways. To enhance the ease of reading and comprehension, this book has been subdivided into various section including; Section I, reviews current progress on our understanding Oncogenic Aspects of HSP90; Section II, focuses on Bimolecular Aspects of HSP90; Section III, emphasizes and HSP90 in Natural Products Development and Section IV; give the most up to date reviews on Clinical Aspects of HSP90. Key basic and clinical research laboratories from major universities, academic medical hospitals, biotechnology and pharmaceutical laboratories around the world have contributed chapters that review present research activity and importantly project the field into the future. The book is a must read for graduate students. medical students, basic science researchers and postdoctoral scholars in the fields of Translational Medicine, Clinical Research, Human Physiology, Biotechnology, Natural Products, Cell & Molecular Medicine, Pharmaceutical Scientists and Researchers involved in Drug Discovery

Expression of h-proIAPP in <i>C</i>. <i>elegans</i> results in protein insolubility and aggregation.

<p>Transgenic h-proIAPP <i>C</i>. <i>elegans</i> model was generated by gonad microinjection of 20 ng/μl of plasmids pPR3, pPR4 and pPR5. Transgenic m-proIAPP <i>C</i>. <i>elegans</i> model was generated by gonad microinjection of 20 ng/μl of plasmids pPR8, pPR9 and pPR10. <b>A.</b> h-proIAPP and m-proIAPP tagged with YFP were expressed in body wall muscles, vulva muscles and anal depressor muscles under <i>lev-11</i> promoter; in pharynx under <i>tnt-4</i> promoter, and in neurons under <i>aex-3</i> promoter. Neuronal expression of proIAPP secreted into the body cavity was observed in the coelomocytes of the h-proIAPP model and distributed in the intestinal region in both h-proIAPP and m-proIAPP <i>C</i>. <i>elegans</i> models. Images were created using maximum intensity projections on all the planes of the acquired stacks except coelomocytes where images were taken in a single plane for better visualization. Arrows indicate areas of fluorescence. DIC images were taken to visualize structures. Results are a representative experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm. <b>B.</b> Data represent the mean fluorescence intensity (MFI) ± SD measured using a sum intensity projection on all the planes of the acquired stacks of m-proIAPP (open bars) and h-proIAPP (filled bars) tagged with YFP expressed in body wall muscles, vulva muscles, anal depressor muscles and pharynx. *p<0.05; **p<0.01 versus m-proIAPP. <b>C.</b> Transgenic h-proIAPP <i>C</i>. <i>elegans</i> tagged with YFP were subjected to FRAP analysis (square). Data are from vulva muscles (row 1), anal depressor (row 2), pharynx (row 3) and coelomocytes (row 4) before FRAP (left panels), 10 seconds after FRAP (middle panels) 250 seconds after FRAP (right panels). Images were obtained using an inverted confocal microscope. Results are representative experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm.</p

Hsp72 expression improves the solubility of h-proIAPP aggregates.

<p><b>A.</b> Transgenic YFP + Hsp72 <i>C</i>. <i>elegans</i> model was generated by co-injection of 20 ng/μl of plasmid pPR18 (that expresses YFP in muscles) together with 30 ng/μl of Hsp72 expressed in the same tissue (plasmid pPR21) to serve as a soluble control (top panels). <i>C</i>. <i>elegans</i> injected with 20 ng/μl of h-proIAPP tagged with YFP expressed in muscles (plasmid pPR3) was used as a control for protein aggregation (middle panels). Transgenic h-proIAPP::YFP + Hsp72 <i>C</i>. <i>elegans</i> animals were generated by gonad co-injection of 20 ng/μl of plasmid pPR3 and 30 ng/μl of plasmid pPR21 (bottom panels). Transgenic animals were subjected to FRAP analysis (square). Data was collected before photobleaching (left panels), 10 seconds after photobleaching (middle panels) and 250 seconds after photobleaching (right panels). Images were obtained using an inverted confocal microscope. Results are representative of one experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm. <b>B.</b> Data represents the quantification of relative fluorescence intensity, RFI ± SEM during recovery after photobleaching of transgenic h-proIAPP::YFP <i>C</i>. <i>elegans</i> model (filled circles), transgenic h-proIAPP::YFP + Hsp72 <i>C</i>. <i>elegans</i> animals (open circles), and transgenic YFP + Hsp72 <i>C</i>. <i>elegans</i> model (filled triangles). Data are the mean of at least three independently performed experiments. *p<0.05 versus h-proIAPP::YFP, Error Bar = SEM.</p