Nutrient and growth factor receptors internalize through dathrin coated pits. The signal sequences which mediate the association between receptors and the coated pit reside in receptor cytoplasmic tail domains. These signal sequences have been extensively investigated in nutrient receptors, and a minimal functional sequence has been identified consisting of a tyrosine residue in an exposed b turn. Protein-protein contacts between internalization signal sequences and components of the coated pit machinery have been proposed to mediate rapid internalization. In vitro evidence suggests the AP-2 adaptor may be that protein component. The signal sequences of growth factor receptors are less well understood. However, a growth factor- and temperature- dependent binding between the epideimal growth factor receptor and the AP-2 adaptor has been observed. We identified Hsc70 as a cytosolic ligand for the cytoplasmic tail of the transferrin receptor. The binding was mapped to the internalization signal sequence of the receptor tail. Mutations within the signal sequence which inhibit internalization result in alteration of signal sequence secondary structure and reduction in stimulation of the Hsc70 ATPase. Co-immunoprecipitation analysis showed a population of transferrin receptors which are bound to Hsc70, suggesting an association in vivo. We also showed binding of Hsc70 to the epidermal growth factor receptor by co-immunoprecipitation analysis. This binding was increased by treatment with EGF. The binding was transient, and occured prior to the binding of the receptor to AP-2 adaptors. Other agents which induce EGF receptor clustering and internalization also stimulate the transient increase in Hsc70 binding and the later AP-2 binding, suggesting a role in early endocytosis. These data support the hypothesis that Hsc70 is associated with the receptors for transferrin and epidermal growth factor in vitro and in vivo. We propose a role for the 70 kD heat shock protein in the assembly/disassembly of protein complexes involved in receptor signalling and/or internalization.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19806号 / 人博第777号 / 新制||人||187(附属図書館) / 27||人博||777(吉田南総合図書館) / 32842 / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 森谷 敏夫, 教授 石原 昭彦 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

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Despite the threat of climate change, the physiological mechanisms by which temperature drives the distribution of species are unclear. Here we used chronic temperature exposures to determine that the upper limit for positive growth in the eastern brook trout (Salvelinus fontinalis) is 23.4 °C. Additionally, brook trout exposed to daily temperature oscillations of 8 °C, around a mean of 21 °C, exhibited growth rates that were 43 and 35% lower by length and weight respectively, than in constant 21 °C controls. Limitations in growth were associated with increases in indicators of the physiological stress response. Individuals exposed to 22 or 24 °C for 24 days exhibited plasma cortisol levels that were 12 and 18 fold greater than at 16 °C. Similarly, gill heat shock protein (Hsp)-70 levels were 10.7 and 56 fold higher at 22 and 24 °C than at 16 °C. Brook trout exposed to daily temperature oscillation of 4 or 8 °C had gill Hsp-70 levels that were 40 and 700 fold greater than controls. Acute (6 h) temperature exposures were used to demonstrate a threshold for induction of the Hsp-70 and plasma glucose responses of 20.7 °C and 21.2 °C respectively. Finally, we conducted field surveys that demonstrated increased plasma cortisol, plasma glucose, and gill Hsp-70 at temperatures above 21 °C. Induction of the cellular and endocrine stress responses is associated with decreased growth in brook trout. Thermal limitations on growth may provide a mechanism by which temperature drives the distributions of this cold-water species.

Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity. As such, mutations in this family of proteins result in a wide variety of diseases from cancers to cardiomyopathies. The sHSPs Beta-2 (HspB2) and alpha-beta crystalline (CryAB) are two of the ten human sHSPs and are both expressed in cardiac and skeletal muscle cells. A heart that cannot properly recover or defend against stressors such as extreme heat or cold, oxidative/reductive stress, and heavy metal-induced stress will constantly struggle to maintain efficient function. Accordingly, CryAB is required for myofibril recovery from ischemia/reperfusion (I/R) and HspB2 is required I/R recovery as well as efficient cardiac ATP production. Despite these critical roles, little is known about the molecular function of these chaperones. We have identified over two hundred HspB2-binding partners through both yeast two-hybrid and copurification approaches, including interactions with myofibril and mitochondrial proteins. There is remarkable overlap between the two approaches (80%) suggesting a high confidence level in our findings. The sHSP, CryAB, only binds a subset of the HspB2 interactome, showing that the HspB2 interactome is specific to HspB2 and supporting non-redundant roles for sHSPs. We have confirmed a subset of these binding partners as HspB2 clients via in vitro chaperone activity assays. In addition, comparing the binding patterns and activity of sHSP variants in comparison to wild type can help to elucidate how variants participate in causing disease. Accordingly, we have used Y2H and in vitro chaperone activity assays to compare the disease-associated human variants R120GCryAB and A177PHspB2 to wild type and have identified differences in binding and chaperone function. These results not only provide the first molecular evidence for non-redundancy of the sHSPs, but provides a useful resource for the study of sHSPs in mitochondrial and myofibril function.

Background: Type 1 Diabetes mellitus (TIDM) is a chronic disorder in which the insulin producing beta cells are selectively self-destroyed. Although the etiology of the disease has not been determined, genetic dispositions such as SLC11A1 polymorphism in suffering patients have been reported. The role of pathogenic microorganisms such as Mycobacterium avium subspecies paratuberculosis (MAP) in TIDM has also been recently debated. MAP is already known to cause paratuberculosis in cattle and now it is a strong suspect of causing autoimmune diseases in humans such as Crohn’s disease, multiple sclerosis, autoimmune Thyroiditis, rheumatoid arthritis and autoimmune diabetes. We hypothesis that molecular mimicry between MAP Heat shock protein 65K (Hsp65) and human Glutamic Acid Decarboxylase 65K (GAD65) can be the trigger which leads to the autoimmune destruction of beta cell in patients exposed to MAP . Method: To test the hypothesis, peptide sequences of MAP Hsp65 and human GAD65 were investigated using BLAST and PyMOL bioinformatics tools. Moreover, 18 blood samples from humans with TIDM and controls, and 100 sera samples from cattle with paratuberculosis and controls were evaluated for the presence of MAP, MAP DNA and its antibodies. Glucose, insulin and GAD65 antibodies were also determined in some of the clinical samples. Results: Peptide BLAST analysis revealed 44% identity between the two proteins with 75% positive identities in a 16 amino acid region. PyMOL structural analysis identified possible shared epitope regions of the proteins in its 3D conformation. Immunoblot analysis revealed a strong cross reactivity between lysate of E.coli recombinant of MAP Hsp65 and plasma from human subject with TIDM. A weak cross reactivity was also observed between healthy rat pancreatic homogenate and rabbit anti MAP IgG. Nested PCR using IS900-specific iv oligonucleotide primers did not detect MAP DNA in peripheral blood from 18 subjects with Type I Diabetes, Type II Diabetes and non-diabetic controls. Long term culture of leukocytes from blood samples from same subjects resulted in the presence of MAP in 3/10 (30%) TIDM and 4/8 (50%) control subjects. However anti MAP IgG were detected in 5/10 (50 %) TIDM samples compared to 3/8 (37.5 %) controls. Insulin level was measured in sera from paratuberculosis cattle and controls. In MAP infected cattle, insulin level ranged from below 0.1ng/ml to 2.456 ng/ml with an average of 0.36 +/- 0.57ng/ml compared to below 0.1ng/ml to 13.47ng/ml with an average of 2.86 +/- 3.00ng/ml in healthy cattle. Conclusion: Bioinformatics analysis between MAP Hsp65 and human GAD65 through BLAST and PyMOL analysis revealed a homology of 16 amino acid motif and possible shared epitope regions; immunohistochemistry analysis revealed a cross reactivity between rabbit antiMAP IgG and pancreatic cell homogenate from a healthy rat. Moreover, plasma from patient with TIDM (TD8), who was confirmed to be positive for MAP DNA and MAP IgG, reacted strongly with MAP Hsp65 in MAP protein lysate and MAP Hsp65 recombinant clone pmptb20. Culture of MAP from human leukocytes is significant despite the lack of correlation between MAP in samples from TIDM and controls. It is worth noting that some of the control subjects have not been evaluated for other autoimmune diseases possible MAP role. Additionally, antiMAP IgG levels in TIDM subjects compared to controls have raised a suspicion on the involvement of MAP in TIDM. The poor correlation of MAP in blood versus either the antiMAP IgG or the insulin level may be related with the fastidious nature of MAP and in vitro cultivation. Since MAP is the sole causative agent of Johne’s disease, it is significant that the insulin level is 8 folds less in MAP infected cattle compared to MAP free cattle. Overall, the data v is mixed and suggest that further study is needed to investigate the intriguing question to whether MAP is involved in TIDM or not.

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