Swapping the linkers of canonical Hsp70 and Hsp110 chaperones compromises both self-association and client selection.

Plasmodium falciparum heat shock protein 70-1 (PfHsp70-1) and PfHsp70-z are essential cytosol localised chaperones of the malaria parasite. The two chaperones functionally interact to drive folding of several parasite proteins. While PfHsp70-1 is regarded as a canonical Hsp70 chaperone, PfHsp70-z belongs to the Hsp110 subcluster. One of the distinctive features of PfHsp70-z is its unique linker segment which delineates it from canonical Hsp70. In the current study, we elucidated the role of the linker in regulating Hsp70 self-association and client selection. Using recombinant forms of PfHsp70-1, PfHsp70-z and E. coli Hsp70 (DnaK) and their respective linker switch mutants we investigated self-association of the chaperones using surface plasmon resonance (SPR) analysis. The effect of the changes on client selectivity was investigated on DnaK and its mutant through co-affinity chromatography coupled to LC-MS analysis. Our findings demonstrated that the linker is important for both Hsp70 self-association and client binding.

Expressions of HSP110 family members in the testes and epididymis of mice at different stages of development and their regulation by hormones / 南方医科大学学报

OBJECTIVE@#To study the expressions of the members of HSP110 family in the testis and epididymis of mice at different stages of development and whether they are regulated by hormones.@*METHODS@#The testicular and epididymis tissues of mice at different ages (14, 21, 28, 35, 42, 49, 70, and 90 days after birth, 3 mice at each age) were collected for RT-PCR detection of the expression levels of HSP110 family members. Forty-eight mice were randomized into 3 groups for sham operation, castration, or castration with testosterone injections every other day (starting at 7 days after castration), and at 1, 3, 5, and 7 days after first testosterone injection, the expressions of HSP110 family in the epididymis were detected using RT-PCR.@*RESULTS@#The mRNA expression levels of HSP110 family members underwent obvious variations with the development of the mice: , and expressions in the testicles of the mice first increased and then decreased, and gradually became stable; they also exhibited similar temporal patterns of changes in the epididymis. In the castrated mice, the mRNA expressions of and in the epididymis decreased significantly with the reduction of serum hormone levels ( < 0.05), and became normal after the supplementation of exogenous hormone.@*CONCLUSIONS@#The expression levels of HSP110 family are affected by developmental regulation, and the expressions of and are under the regulation by hormones.

Expression and function of HSP110 family in mouse testis after vasectomy / 亚洲男科学杂志(英文版)

HSP110 functions to protect cells, tissues, and organs from noxious conditions. Vasectomy induces apoptosis in the testis; however, little is known about the reason leading to this outcome. The aim of the present study was to evaluate the expression and function of HSP110 in mouse testis after vasectomy. Following bilateral vasectomy, we used fluorescent Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to detect apoptosis, Western blotting and immunohistochemistry to examine HSP110 expression and localization. Serum antisperm antibody (AsAb) and testosterone were measured by Enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. Expression of endoplasmic reticulum stress (ERS) sensors and downstream signaling components was measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and the phosphorylation of eIF2 and JNK was detected by Western blotting. Vasectomy induced morphologic changes, increased apoptosis in the testis, increased serum AsAb, and decreased testosterone levels. After vasectomy, ORP150 mRNA level was increased first and then decreased, Bcl-2 was decreased, and the expression of HSPA4l, GRP78, GADD153, PERK, ATF6, IRE-1, XBP-1s, Bax, Bak, and caspases and the phosphorylation of eIF2 and JNK were increased. We present that an ER stress-mediated pathway is activated and involved in apoptosis in the testis after vasectomy. HSPA4l and ORP150 may play important roles in maintaining the normal structure and function of testis.

Heat shock protein 110 improves the immunological effect of an altered peptide ligand of human papilloma virus type 16 E711-20 peptide / 中华皮肤科杂志

ObjectiveTo investigate the adjuvant effect of heatshock protein 110(HSP110) on the immune responses induced by an altered peptide ligand of human papilloma virus type 16 E711-20 peptide (HPV16E711-20).Methods The complex of HSP110 and an altered peptide ligand of HPV16E711-20 was constructed in vitro.Fifteen 6-week-old C57BL/6 female mice were randonly and equally divided into 3 groups,including complex group,ligand group,and phosphate buffered solution (PBS) group,to receive intraperitoneal immunization with the complex (100 μg),peptide (10 μg),and PBS (100 μl) respectively.Immunization was carried out with an interval of 2 weeks for 2 times.Two weeks after the last immunization,the mice were sacrificed followed by the isolation of splenocytes.MTT assay was performed to evaluate the proliferation activity of splenocytes,intracellular staining for interferon(INF)-γ to detect cytotoxic T lymphocytes (CTLs),standard chromium-51 (51Cr) release assay to estimate the lethal effect of specific CTLs on target cells.Statistical analysis was performed by using t test with SPSS 10.0 software.Differences were considered as statistically significant when the P value was less than 0.05.ResultsA significant increase was observed in the proliferation index ( 1.87 ± 0.122 vs.0.32 ± 0.071,t =4.01,P ＜ 0.01 ) of,and percentage of CD8+IFN-γ+ T lymphocytes(3.9％ vs.0.4％,t =3.88,P ＜ 0.01 ) among splenocytes from the complex group compared with the ligand group.At the effector-to-target ratio of 100 ∶ 1,50 ∶ 1,25∶ 1 and 12.5 ∶ 1,the death rate of target cells was 54.7％,72.2％,61.5％ and 39.8％ respectively after incubation with CTLs from the compleximmunized mice,higher than that from the ligand-immunized mice (35.2％,49.3％,28.1％,17.4％,respectively).ConclusionHSP110 could enhance the immunological effect of the altered peptide ligand of HPV16E711-20,and can serve as an immunological adjuvant.

Immunogenicity of the immunodominant cytotoxic T lymphocyte epitope E749-57 in HPV16 oncoprotein E7 chaperoned by HSP110 / 中华皮肤科杂志

Objective To investigate the immunogenicity of immunodominant cytotoxic T lymphocyte epitope E749-57 of human papilloma virus (HPV) 16 oncoprotein E7 chaperoned by heat shock protein (HSP)110. Methods Mouse HSP110 gene was cloned into prokaryotic expression vector pQE-80L for the expression of HSP110 protein, which was purified using Ni-NTA column. SDS-PAGE and Western-blot were conducted to confirm the purified mHSP110 protein, which was subsequently incubated with E749-57 peptide under heat shock condition, and high-performance liquid chromatography (HPLC) was used to evaluate the binding efficiency of the recombinant protein and E749-57 peptide. Twenty mice were divided into 4 groups to be immunized with mHSP110 protein, E749-57 peptide, mHSP110-E749-57 complex and phosphate buffered saline (PBS),respectively. Two weeks after the last immunization, spleen cells were collected from the immunized mice and divided into 2 parts: one were stimulated by E749-57 peptide followed by the detection of CD8+ INF-γ+ T cells with flow cytometry; the other one were subjected to MTT analysis for the estimation of cell proliferation. The mHSP110-E749-57 complex was also used to immunize TC-1 tumor bearing mice to observe its anti-tumor effect.Results The full-length 2577 bp-sized mHSP110 gene was amplified from mouse liver cDNA and cloned into pQE-80L vector. Direct sequencing confirmed the correctness of the cloning. SDS-PAGE and Western-blot demonstrated the successful purification of mHSP110. HPLC assay showed that the purified mHSP110 protein could bind with E749-57 to form a relatively stable protein complex. The percentage of IFN-γ+ CD8+ T cells in and proliferation index of spleen cells from the complex-immunized mice were statistically higher than those from the other 3 groups of mice. Moreover, the complex could obviously inhibit the growth of TC-1 tumor in mice. Conclusion The mHSP110-E749-57 complex could enhance the generation of specific cytotoxic T lymphocytes and exert anti-tumor effects in mice.