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Supporting data on prion protein translocation mechanism revealed by pulling force studies.

ABSTRACT: The Prion protein (PrP) is a highly conserved cell surface glycoprotein. To enter the secretory pathway, the PrP precursor relies on the Sec61 complex and multiple accessory factors all gathering at the membrane of the Endoplasmic reticulum (ER). PrP topogenesis results in the formation of different PrP isoforms. Aside from the typical secretory variant (^SecPrP) different pathognomonic, membrane-embedded variants (^NtmPrP and ^CtmPrP) that are associated with neurodegenerative diseases can be found [1]. In this article, we provide supportive data related to "Prion Protein Translocation Mechanism Revealed by Pulling Force Studies" (Kriegler et al., May 2020)[2], where we utilize Xbp1 arrest peptide (AP)-mediated ribosomal stalling to study the co-translational folding experienced by PrP during its insertion into the ER. We measure translocation efficiency and characterize the force exerted on PrP nascent chain so called "pulling force profile". Here, we describe the method of AP-mediated ribosomal stalling assay together with additional experimental data to the main article. Furthermore, we describe the combination of AP-mediated ribosomal stalling and semi-permeabilized Hela cells (SPCs) as ER membrane source. Using this experimental set-up one can directly determine the contribution of a specific membrane component, e.g. subunits of the ER protein translocase, as pulling factor exerting force on the PrP nascent chain. The data presented here covers (a) the SDS-PAGE gel images visualized by autoradiography, (b) quantification of the different populations of PrP species observed in the AP-mediated ribosomal stalling method, and (c) calculation formulas of the pulling force profiles measured in SPCs in comparison to dog pancreas microsomes as ER membrane donor. Finally, Western Blot analysis and quantification of siRNA knockdown levels compared to control conditions of various translocation components are shown.

SUBMITTER: Kriegler T

PROVIDER: S-EPMC7334574 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We have an operant rat model of upper extremity reaching and grasping in which we examined the impact of performing a high force high repetition (High-ForceHR) versus a low force low repetition (Low-ForceHR) task for 18weeks on the radius and ulna, compared to age-matched controls. High-ForceHR rats performed at 4 reaches/min and 50% of their maximum voluntary pulling force for 2h/day, 3days/week. Low-ForceHR rats performed at 6% maximum voluntary pulling force. High-ForceHR rats showed decreased trabecular bone volume in the distal metaphyseal radius, decreased anabolic indices in this same bone region (e.g., decreased osteoblasts and bone formation rate), and increased catabolic indices (e.g., microcracks, increased osteocyte apoptosis, secreted sclerostin, RANKL, and osteoclast numbers), compared to controls. Distal metaphyseal trabeculae in the ulna of High-ForceHR rats showed a non-significant decrease in bone volume, some catabolic indices (e.g., decreased trabecular numbers) yet also some anabolic indices (e.g., increased osteoblasts and trabecular thickness). In contrast, the mid-diaphyseal region of High-ForceHR rats' radial and ulnar bones showed few to no microarchitecture differences and no changes in apoptosis, sclerostin or RANKL levels, compared to controls. In further contrast, Low-ForceHR rats showed increased trabecular bone volume in the radius in the distal metaphysis and increased cortical bone area its mid-diaphysis. These changes were accompanied by increased anabolic indices, no microcracks or osteocyte apoptosis, and decreased RANKL in each region, compared to controls. Ulnar bones of Low-ForceHR rats also showed increased anabolic indices, although fewer than in the adjacent radius. Thus, prolonged performance of an upper extremity reaching and grasping task is loading-, region-, and bone-dependent, with high force loads at high repetition rates inducing region-specific increases in bone degradative changes that were most prominent in distal radial trabeculae, while low force task loads at high repetition rates induced adaptive bone responses.

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Supporting data on prion protein translocation mechanism revealed by pulling force studies.

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