Prion Protein Folding Mechanism Revealed by Pulling Force Studies

AbstractThe 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 a 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 topological complex protein, where marginal pulling by the signal sequence, together with the downstream sequence in the mature domain, primarily drives an overall inefficient translocation resulting in the nascent chain to adopt other topologies.

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Cotranslational Partitioning of Nascent Prion Protein into Multiple Populations at the Translocation Channel

Molecular Biology of the Cell ◽

10.1091/mbc.e02-05-0293 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3775-3786 ◽

Cited By ~ 60

Author(s):

Soo Jung Kim ◽

Ramanujan S. Hegde

Keyword(s):

Endoplasmic Reticulum ◽

Prion Protein ◽

Mechanism Of Action ◽

Transmembrane Domain ◽

Signal Sequence ◽

Multiple Populations ◽

Sequence Of Events ◽

N Terminus ◽

Complete Reversal ◽

Single Polypeptide

The decisive events that direct a single polypeptide such as the prion protein (PrP) to be synthesized at the endoplasmic reticulum in both fully translocated and transmembrane forms are poorly understood. In this study, we demonstrate that the topological heterogeneity of PrP is determined cotranslationally, while at the translocation channel. By evaluating sequential intermediates during PrP topogenesis, we find that signal sequence-mediated initiation of translocation results in an interaction between nascent PrP and endoplasmic reticulum chaperones, committing the N terminus to the lumen. Synthesis of the transmembrane domain before completion of this step allows it to direct the generation of CtmPrP, a transmembrane form with its N terminus in the cytosol. Thus, segregation of nascent PrP into different topological configurations is critically dependent on the precise timing of signal-mediated initiation of N-terminus translocation. Consequently, this step could be experimentally tuned to modify PrP topogenesis, including complete reversal of the elevatedCtmPrP caused by disease-associated mutations in the transmembrane domain. These results delineate the sequence of events involved in PrP biogenesis, explain the mechanism of action ofCtmPrP-favoring mutations associated with neurodegenerative disease, and more generally, reveal that translocation substrates can be cotranslationally partitioned into multiple populations at the translocon.

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Protein Targeting to the Bacterial Cytoplasmic Membrane

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.63.1.161-173.1999 ◽

1999 ◽

Vol 63 (1) ◽

pp. 161-173 ◽

Cited By ~ 170

Author(s):

Peter Fekkes ◽

Arnold J. M. Driessen

Keyword(s):

Protein Targeting ◽

Cytoplasmic Membrane ◽

Signal Sequence ◽

Specific Interaction ◽

Signal Recognition Particle ◽

High Fidelity ◽

Signal Recognition ◽

Nascent Chain ◽

Major Route ◽

Mature Domain

SUMMARY Proteins that perform their activity within the cytoplasmic membrane or outside this cell boundary must be targeted to the translocation site prior to their insertion and/or translocation. In bacteria, several targeting routes are known; the SecB- and the signal recognition particle-dependent pathways are the best characterized. Recently, evidence for the existence of a third major route, the twin-Arg pathway, was gathered. Proteins that use either one of these three different pathways possess special features that enable their specific interaction with the components of the targeting routes. Such targeting information is often contained in an N-terminal extension, the signal sequence, but can also be found within the mature domain of the targeted protein. Once the nascent chain starts to emerge from the ribosome, competition for the protein between different targeting factors begins. After recognition and binding, the targeting factor delivers the protein to the translocation sites at the cytoplasmic membrane. Only by means of a specific interaction between the targeting component and its receptor is the cargo released for further processing and translocation. This mechanism ensures the high-fidelity targeting of premembrane and membrane proteins to the translocation site.

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Faculty Opinions recommendation of Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.2183956.2177101 ◽

2010 ◽

Author(s):

Joachim Seemann

Keyword(s):

Prion Protein ◽

Signal Sequence

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PrP P102L and Nearby Lysine Mutations Promote Spontaneous In Vitro Formation of Transmissible Prions

Journal of Virology ◽

10.1128/jvi.01276-17 ◽

2017 ◽

Vol 91 (21) ◽

Cited By ~ 6

Author(s):

Allison Kraus ◽

Gregory J. Raymond ◽

Brent Race ◽

Katrina J. Campbell ◽

Andrew G. Hughson ◽

...

Keyword(s):

Prion Protein ◽

Prion Diseases ◽

Transmissible Spongiform Encephalopathy ◽

Clinical Signs ◽

Protein Aggregates ◽

Structural Features ◽

Spongiform Encephalopathy ◽

Specific Sequence

ABSTRACT Accumulation of fibrillar protein aggregates is a hallmark of many diseases. While numerous proteins form fibrils by prion-like seeded polymerization in vitro, only some are transmissible and pathogenic in vivo. To probe the structural features that confer transmissibility to prion protein (PrP) fibrils, we have analyzed synthetic PrP amyloids with or without the human prion disease-associated P102L mutation. The formation of infectious prions from PrP molecules in vitro has required cofactors and/or unphysiological denaturing conditions. Here, we demonstrate that, under physiologically compatible conditions without cofactors, the P102L mutation in recombinant hamster PrP promoted prion formation when seeded by minute amounts of scrapie prions in vitro. Surprisingly, combination of the P102L mutation with charge-neutralizing substitutions of four nearby lysines promoted spontaneous prion formation. When inoculated into hamsters, both of these types of synthetic prions initiated substantial accumulation of prion seeding activity and protease-resistant PrP without transmissible spongiform encephalopathy (TSE) clinical signs or notable glial activation. Our evidence suggests that PrP's centrally located proline and lysine residues act as conformational switches in the in vitro formation of transmissible PrP amyloids. IMPORTANCE Many diseases involve the damaging accumulation of specific misfolded proteins in thread-like aggregates. These threads (fibrils) are capable of growing on the ends by seeding the refolding and incorporation of the normal form of the given protein. In many cases such aggregates can be infectious and propagate like prions when transmitted from one individual host to another. Some transmitted aggregates can cause fatal disease, as with human iatrogenic prion diseases, while other aggregates appear to be relatively innocuous. The factors that distinguish infectious and pathogenic protein aggregates from more innocuous ones are poorly understood. Here we have compared the combined effects of prion seeding and mutations of prion protein (PrP) on the structure and transmission properties of synthetic PrP aggregates. Our results highlight the influence of specific sequence features in the normally unstructured region of PrP that influence the infectious and neuropathogenic properties of PrP-derived aggregates.

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Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein

The Journal of Cell Biology ◽

10.1083/jcb.200911115 ◽

2010 ◽

Vol 188 (4) ◽

pp. 515-526 ◽

Cited By ~ 50

Author(s):

Neena S. Rane ◽

Oishee Chakrabarti ◽

Lionel Feigenbaum ◽

Ramanujan S. Hegde

Keyword(s):

Prion Protein ◽

Protein Translocation ◽

Signal Sequence ◽

Signal Sequences ◽

Hydrophobic Domain ◽

Sequence Variations ◽

Sufficient Magnitude ◽

The Impact

Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations.

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Stage- and ribosome-specific alterations in nascent chain-Sec61p interactions accompany translocation across the ER membrane.

The Journal of Cell Biology ◽

10.1083/jcb.129.4.957 ◽

1995 ◽

Vol 129 (4) ◽

pp. 957-970 ◽

Cited By ~ 27

Author(s):

C V Nicchitta ◽

E C Murphy ◽

R Haynes ◽

G S Shelness

Keyword(s):

Protein Translocation ◽

Signal Sequence ◽

Near Neighbor ◽

Cross Linking ◽

Dramatic Reduction ◽

Wild Type ◽

Early Stages ◽

Nascent Chain ◽

Chemical Cross Linking ◽

Er Membrane

Near-neighbor interactions between translocating nascent chains and Sec61p were investigated by chemical cross-linking. At stages of translocation before signal sequence cleavage, nascent chains could be cross-linked to Sec61p at high (60-80%) efficiencies. Cross-linking occurred through the signal sequence and the mature portion of wild-type and signal cleavage mutant nascent chains. At later stages of translocation, as represented through truncated translocation intermediates, cross-linking to Sec61p was markedly reduced. Dissociation of the ribosome into its large and small subunits after assembly of the precursor into the translocon, but before cross-linking, resulted in a dramatic reduction in subsequent cross-linking yield, indicating that at early stages of translocation, nascent chain-Sec61p interactions are in part mediated through interactions of the ribosome with components of the ER membrane, such as Sec61p. Dissociation of the ribosome was, however, without effect on subsequent translocation. These results are discussed with respect to a model in which Sec61p performs a function essential for the initiation of protein translocation.

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Real-time observation of signal recognition particle binding to actively translating ribosomes

eLife ◽

10.7554/elife.04418 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 28

Author(s):

Thomas R Noriega ◽

Jin Chen ◽

Peter Walter ◽

Joseph D Puglisi

Keyword(s):

Real Time ◽

Single Molecule ◽

Protein Translocation ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Initial Step ◽

Signal Recognition ◽

Fluorescence Measurements ◽

Nascent Chain ◽

Time Observation

The signal recognition particle (SRP) directs translating ribosome-nascent chain complexes (RNCs) that display a signal sequence to protein translocation channels in target membranes. All previous work on the initial step of the targeting reaction, when SRP binds to RNCs, used stalled and non-translating RNCs. This meant that an important dimension of the co-translational process remained unstudied. We apply single-molecule fluorescence measurements to observe directly and in real-time E. coli SRP binding to actively translating RNCs. We show at physiologically relevant SRP concentrations that SRP-RNC association and dissociation rates depend on nascent chain length and the exposure of a functional signal sequence outside the ribosome. Our results resolve a long-standing question: how can a limited, sub-stoichiometric pool of cellular SRP effectively distinguish RNCs displaying a signal sequence from those that are not? The answer is strikingly simple: as originally proposed, SRP only stably engages translating RNCs exposing a functional signal sequence.

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From folding to function: complex macromolecular reactions unraveled one-by-one with optical tweezers

Essays in Biochemistry ◽

10.1042/ebc20200024 ◽

2021 ◽

Author(s):

Pétur O. Heidarsson ◽

Ciro Cecconi

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Structural Changes ◽

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Disordered Proteins ◽

Kinase Activation ◽

Intrinsically Disordered ◽

Complex Protein ◽

Methodological Principles

Abstract Single-molecule manipulation with optical tweezers has uncovered macromolecular behaviour hidden to other experimental techniques. Recent instrumental improvements have made it possible to expand the range of systems accessible to optical tweezers. Beyond focusing on the folding and structural changes of isolated single molecules, optical tweezers studies have evolved into unraveling the basic principles of complex molecular processes such as co-translational folding on the ribosome, kinase activation dynamics, ligand–receptor binding, chaperone-assisted protein folding, and even dynamics of intrinsically disordered proteins (IDPs). In this mini-review, we illustrate the methodological principles of optical tweezers before highlighting recent advances in studying complex protein conformational dynamics – from protein synthesis to physiological function – as well as emerging future issues that are beginning to be addressed with novel approaches.

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An Empirical Analysis of Test Input Generation Tools for Android Apps through a Sequence of Events

Symmetry ◽

10.3390/sym12111894 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1894

Author(s):

Husam N. Yasin ◽

Siti Hafizah Ab Hamid ◽

Raja Jamilah Raja Yusof ◽

Muzaffar Hamzah

Keyword(s):

Computational Cost ◽

Sequence Length ◽

Code Coverage ◽

Android Apps ◽

Gui Testing ◽

Test Input ◽

Testing Tools ◽

Sequence Of Events ◽

Current State ◽

A Minor

Graphical User Interface (GUI) testing of Android apps has gained considerable interest from the industries and research community due to its excellent capability to verify the operational requirements of GUI components. To date, most of the existing GUI testing tools for Android apps are capable of generating test inputs by using different approaches and improve the Android apps’ code coverage and fault detection performance. Many previous studies have evaluated the code coverage and crash detection performances of GUI testing tools in the literature. However, very few studies have investigated the effectiveness of the test input generation tools, especially in the events sequence length of the overall test coverage and crash detection. The event sequence length generally shows the number of steps required by the test input generation tools to detect a crash. It is critical to highlight its effectiveness due to its significant effects on time, testing effort, and computational cost. Thus, this study evaluated the effectiveness of six test input generation tools for Android apps that support the system events generation on 50 Android apps. The generation tools were evaluated and compared based on the activity coverage, method coverage, and capability in detecting crashes. Through a critical analysis of the results, this study identifies the diversity and similarity of test input generation tools for Android apps to provide a clear picture of the current state of the art. The results revealed that a long events sequence performed better than a shorter events sequence. However, a long events sequence led to a minor positive effect on the coverage and crash detection. Moreover, the study showed that the tools achieved less than 40% of the method coverage and 67% of the activity coverage.

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