Heteromeric complexes of heat shock protein 70 (HSP70) family members, including Hsp70B', in differentiated human neuronal cells

Abstract

Human neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have been termed "protein misfolding disorders." Upregulation of heat shock proteins that target misfolded aggregation-prone proteins has been proposed as a potential therapeutic strategy to counter neurodegenerative disorders. The heat shock protein 70 (HSP70) family is well characterized for its cytoprotective effects against cell death and has been implicated in neuroprotection by overexpression studies. HSP70 family members exhibit sequence and structural conservation. The significance of the multiplicity of HSP70 proteins is unknown. In this study, coimmunoprecipitation was employed to determine if association of HSP70 family members occurs, including Hsp70B' which is present in the human genome but not in mouse and rat. Heteromeric complexes of Hsp70B', Hsp70, and Hsc70 were detected in differentiated human SH-SY5Y neuronal cells. Hsp70B' also formed complexes with Hsp40 suggesting a common co-chaperone for HSP70 family members.

Fig. 1

Expression of HSP70 family proteins in differentiated human neuronal cells following treatment with celastrol. Differentiated human SH-SY5Y neuronal cells were treated with either 0.75 µM of celastrol (Ce), or heat shock (HS) at 43ºC for 10 min, and allowed to recover at 37ºC for time points up to 48 h. Robust and sustained expression of Hsp70 and Hsp70B′ was induced by celastrol, but not by heat shock. Levels of constitutive Hsc70 showed little change following either celastrol or heat shock treatments. β-tubulin was used as a loading control. Antibodies employed for the detection of HSP70 family proteins: Hsp70 (SPA-810), Hsp70B′ (SPA-754), and Hsc70 (SPA-815). DMSO, Cells treated with DMSO, the celastrol vehicle; C, cells receiving no DMSO or celastrol

Fig. 2

Specificity of antibodies used to detect HSP70 family proteins. Purified recombinant human proteins (0.5 µg) of Hsp70, Hsc70, and Hsp70B′ were subjected to Western blot analysis. a For the detection of Hsp70B′ and Hsc70, antibodies SPA-754 and SPA-815 were found to be specific. b Screening of Hsp70 antibodies was performed. Commercially available anti-Hsp70 antibodies either cross-reacted with Hsp70B′ (SPA-812, 3096, and the commonly used SPA-810) or failed to detect a signal (ab45133, 554243)

Fig. 3

Heteromeric complexes of HSP70 family proteins Hsp70B′, Hsp70, and Hsc70 in differentiated human neuronal cells. a Co-IP using the Hsp70B′-specific antibody demonstrated complex formation of Hsp70B′ with Hsp70 and Hsc70. b Co-IP of Hsc70 showed association of Hsc70 with Hsp70B′ and Hsp70. c Co-IP of Hsp70 suggested its association with Hsp70B′ and Hsc70. IP, antibody used for Co-IP; Western, antibody used for immunodetection of Co-IP pull-down product

Fig. 4

Co-immunoprecipitation of YFP–Hsp70 fusion protein with Hsp70B′ and Hsc70. a A human SH-SY5Y neuronal cell line stably expressing YFP–Hsp70 fusion protein was generated to bypass the need for the use of an anti-Hsp70 antibody in the Co-IP assay. Stable expression of YFP–Hsp70 fusion protein was detected with an anti-YFP antibody and by the increased molecular weight of the signal. β-tubulin was used as a loading control. b Co-IP was performed with the YFP-specific antibody. Immunodetection with YFP antibody demonstrated successful pull-down of YFP–Hsp70 fusion protein. Association of Hsp70B′ and Hsc70 with the YFP–Hsp70 fusion protein was detected in the pull-down products

Fig. 5

Association of Hsp70B′ with Hsp40. a Constitutive expression of Hsp40 was detected in differentiated human neuronal cells under all conditions examined (DMSO, cells treated with DMSO, the celastrol vehicle; C, cells receiving no DMSO or celastrol; Ce, cells treated with celastrol at 0.75 µM; HS, cells heat shocked at 43ºC for 10 min). β-tubulin was used as a loading control. b Co-IP of Hsp70B′ demonstrated its association with Hsp40