| 1. |
- Kriegler, Theresa, et al.
(author)
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Prion Protein Translocation Mechanism Revealed by Pulling Force Studies
- 2020
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In: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 432:16, s. 4447-4465
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Journal article (peer-reviewed)abstract
- The mammalian prion protein (PrP) engages with the ribosome-Sec61 translocation channel complex to generate different topological variants that are either physiological, or involved in neurodegenerative diseases. Here, we describe cotranslational folding and translocation mechanisms of PrP coupled to an Xbp1-based arrest peptide (AP) as folding sensor, to measure forces acting on PrP nascent chain. Our data reveal two main pulling events followed by a minor third one exerted on the nascent chains during their translocation. Using those force landscapes, we show that a specific sequence within an intrinsically disordered region, containing a polybasic and glycine-proline rich residues, modulates the second pulling event by interacting with TRAP complex. This work also delineates the sequence of events involved in generation of PrP toxic transmembrane topologies during its synthesis. Our results shed new insight into the folding of such a topological complex protein, where marginal pulling by the signal sequence, together with the flanking downstream sequence in the mature domain, primarily drives an overall inefficient translocation resulting in the nascent chain to adopt alternative topologies.
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| 2. |
- Kriegler, Theresa, et al.
(author)
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Supporting data on prion protein translocation mechanism revealed by pulling force studies
- 2020
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In: Data in Brief. - : Elsevier BV. - 2352-3409. ; 31
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Journal article (peer-reviewed)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.
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| 3. |
- Notari, Luigi, et al.
(author)
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Cotranslational Folding of a Pentarepeat beta-Helix Protein
- 2018
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In: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 430:24, s. 5196-5206
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Journal article (peer-reviewed)abstract
- It is becoming increasingly clear that many proteins start to fold cotranslationally before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, that is, proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a beta-helix pentarepeat protein from the human pathogen Clostridium botulinum a homolog of the fluoroquinolone resistance protein MfpA-using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight beta-helix coils in the protein produces enough force to release ribosome stalling and that folding starts when this unit is similar to 35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo and may reflect the minimal size of a stable beta-helix since it is structurally homologous to the smallest known beta-helix protein, a four-coil protein that is stable in solution.
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