Passage Practice: Amino Acids, Peptides, and Proteins

Prion diseases are incurable neurological disorders that produce a broad range of symptoms in mammalian species including humans (Creutzfeldt–Jacob Disease (CJD), Gerstmann–Sträussler–Scheinker (GSS), Fatal Familial Insomnia (FFI), Kuru) and cattle (Bovine Spongiform Encephalopathy (BSE)).

Prion diseases are characterized by the misfolding of a normal protein (cellular prion protein, PrPC) into the pathological β-sheet-rich isoform defined scrapie prion protein (PrPSc), which represents an essential component in the pathophysiology of neurodegenerative prion diseases whose etiology can be infectious, sporadic or genetic.

In the case of infectious prion diseases, the formation of nascent prions has been proposed to be driven by a direct interaction between the pathogenic PrPSc template and the endogenous PrPC substrate. By contrast, in the genetic forms, alteration in PrPC conformation may be induced by a genetic mutation in Prnp gene encoding PrPC.

PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein located on the cell surface in lipid–enriched microdomains. Interestingly, contrasting data indicate that (i) the lipid and protein environment at the plasma membrane might be favourable for PrPC–PrPSc interaction and conversion or that (ii) they can have a protective role in pathological scrapie conversion of PrP mutants.

Some mutations leading to genetic prion diseases, characterized by PrPSc accumulation, are not only present in the C-terminal domain of PrPC but are also present in the GPI-attachment signal, implying that the GPI-anchor signal itself can also play a role in neurodegeneration. The GPI-anchor remodelling steps through the passage to the ER and Golgi (critical cellular organelles for chaperoning folding processes) are essential for the final protein localization in the plasma membrane, which in turn together with endosomal recycling compartment, has been considered to participate in PrPSc conversion.

Adapted From:

Sarnataro, D. Attempt to Untangle the Prion-Like Misfolding Mechanism for Neurodegenerative Diseases. Int. J. Mol. Sci. 2018, 19, 3081. https://doi.org/10.3390/ijms19103081

Prion folding diseases such as CJD are caused by an alteration in what element of a protein’s structure?

Prion diseases are incurable neurological disorders that produce a broad range of symptoms in mammalian species including humans (Creutzfeldt–Jacob Disease (CJD), Gerstmann–Sträussler–Scheinker (GSS), Fatal Familial Insomnia (FFI), Kuru) and cattle (Bovine Spongiform Encephalopathy (BSE)).

Prion diseases are characterized by the misfolding of a normal protein (cellular prion protein, PrPC) into the pathological β-sheet-rich isoform defined scrapie prion protein (PrPSc), which represents an essential component in the pathophysiology of neurodegenerative prion diseases whose etiology can be infectious, sporadic or genetic.

In the case of infectious prion diseases, the formation of nascent prions has been proposed to be driven by a direct interaction between the pathogenic PrPSc template and the endogenous PrPC substrate. By contrast, in the genetic forms, alteration in PrPC conformation may be induced by a genetic mutation in Prnp gene encoding PrPC.

PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein located on the cell surface in lipid–enriched microdomains. Interestingly, contrasting data indicate that (i) the lipid and protein environment at the plasma membrane might be favourable for PrPC–PrPSc interaction and conversion or that (ii) they can have a protective role in pathological scrapie conversion of PrP mutants.

Some mutations leading to genetic prion diseases, characterized by PrPSc accumulation, are not only present in the C-terminal domain of PrPC but are also present in the GPI-attachment signal, implying that the GPI-anchor signal itself can also play a role in neurodegeneration. The GPI-anchor remodelling steps through the passage to the ER and Golgi (critical cellular organelles for chaperoning folding processes) are essential for the final protein localization in the plasma membrane, which in turn together with endosomal recycling compartment, has been considered to participate in PrPSc conversion.

Adapted From:

Sarnataro, D. Attempt to Untangle the Prion-Like Misfolding Mechanism for Neurodegenerative Diseases. Int. J. Mol. Sci. 2018, 19, 3081. https://doi.org/10.3390/ijms19103081

PrpC and their GPI anchors are most likely to be associated with which of the following membrane elements?

Prion diseases are incurable neurological disorders that produce a broad range of symptoms in mammalian species including humans (Creutzfeldt–Jacob Disease (CJD), Gerstmann–Sträussler–Scheinker (GSS), Fatal Familial Insomnia (FFI), Kuru) and cattle (Bovine Spongiform Encephalopathy (BSE)).

Prion diseases are characterized by the misfolding of a normal protein (cellular prion protein, PrPC) into the pathological β-sheet-rich isoform defined scrapie prion protein (PrPSc), which represents an essential component in the pathophysiology of neurodegenerative prion diseases whose etiology can be infectious, sporadic or genetic.

In the case of infectious prion diseases, the formation of nascent prions has been proposed to be driven by a direct interaction between the pathogenic PrPSc template and the endogenous PrPC substrate. By contrast, in the genetic forms, alteration in PrPC conformation may be induced by a genetic mutation in Prnp gene encoding PrPC.

PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein located on the cell surface in lipid–enriched microdomains. Interestingly, contrasting data indicate that (i) the lipid and protein environment at the plasma membrane might be favourable for PrPC–PrPSc interaction and conversion or that (ii) they can have a protective role in pathological scrapie conversion of PrP mutants.

Some mutations leading to genetic prion diseases, characterized by PrPSc accumulation, are not only present in the C-terminal domain of PrPC but are also present in the GPI-attachment signal, implying that the GPI-anchor signal itself can also play a role in neurodegeneration. The GPI-anchor remodelling steps through the passage to the ER and Golgi (critical cellular organelles for chaperoning folding processes) are essential for the final protein localization in the plasma membrane, which in turn together with endosomal recycling compartment, has been considered to participate in PrPSc conversion.

Adapted From:

Sarnataro, D. Attempt to Untangle the Prion-Like Misfolding Mechanism for Neurodegenerative Diseases. Int. J. Mol. Sci. 2018, 19, 3081. https://doi.org/10.3390/ijms19103081

According to the passage changes in which of the following are seen first in infectious prion diseases and hereditary prion diseases?

Prion diseases are incurable neurological disorders that produce a broad range of symptoms in mammalian species including humans (Creutzfeldt–Jacob Disease (CJD), Gerstmann–Sträussler–Scheinker (GSS), Fatal Familial Insomnia (FFI), Kuru) and cattle (Bovine Spongiform Encephalopathy (BSE)).

Prion diseases are characterized by the misfolding of a normal protein (cellular prion protein, PrPC) into the pathological β-sheet-rich isoform defined scrapie prion protein (PrPSc), which represents an essential component in the pathophysiology of neurodegenerative prion diseases whose etiology can be infectious, sporadic or genetic.

In the case of infectious prion diseases, the formation of nascent prions has been proposed to be driven by a direct interaction between the pathogenic PrPSc template and the endogenous PrPC substrate. By contrast, in the genetic forms, alteration in PrPC conformation may be induced by a genetic mutation in Prnp gene encoding PrPC.

PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein located on the cell surface in lipid–enriched microdomains. Interestingly, contrasting data indicate that (i) the lipid and protein environment at the plasma membrane might be favourable for PrPC–PrPSc interaction and conversion or that (ii) they can have a protective role in pathological scrapie conversion of PrP mutants.

Some mutations leading to genetic prion diseases, characterized by PrPSc accumulation, are not only present in the C-terminal domain of PrPC but are also present in the GPI-attachment signal, implying that the GPI-anchor signal itself can also play a role in neurodegeneration. The GPI-anchor remodelling steps through the passage to the ER and Golgi (critical cellular organelles for chaperoning folding processes) are essential for the final protein localization in the plasma membrane, which in turn together with endosomal recycling compartment, has been considered to participate in PrPSc conversion.

Adapted From:

Sarnataro, D. Attempt to Untangle the Prion-Like Misfolding Mechanism for Neurodegenerative Diseases. Int. J. Mol. Sci. 2018, 19, 3081. https://doi.org/10.3390/ijms19103081

The Golgi and ER play a direct role in what element GPI signal remodeling?

Amyloids are unbranched protein fibrils in which monomers form intermolecular β-sheets stabilized by numerous hydrogen bonds. This structure, called cross-β, gives amyloids unusual resistance to treatment with ionic detergents (such as SDS and sarkosyl). Initially, amyloids were described as the lethal pathogenic agents of incurable diseases called amyloidoses in humans. In recent decades, it has become clear that amyloids are not only involved in pathogenesis; they also play an essential role in a wide spectrum of biological processes.

A proteomic method called PSIA (Proteomic Screening and Identification of Amyloids) allows for the identification of candidates for amyloid-forming proteins in the complete proteomes of different organisms, was recently developed. Using PSIA scientists analyzed YghJ a secreted protein harboring an evolutionarily conserved zinc metalloprotease domain called M60 in E coli.

To analyze the properties of YghJM aggregation, scientists used a plasmid encoding YghJM C-terminally tagged with His6 and under the control of an IPTG-inducible T7 promoter. YghJM was then purified using column chromatography from E. coli and then transferred into non-denaturing conditions to allow spontaneous aggregation. These aggregates were then subjected to PSIA and the M60-like metalloprotease domain of YghJ was found to have a high amyloidogenic propensity: comprised of 3 individual amyloidogenic regions.

Adapted From:

Belousov MV, Bondarev SA, Kosolapova AO, Antonets KS, Sulatskaya AI, Sulatsky MI, et al. (2018) M60-like metalloprotease domain of the Escherichia coli YghJ protein forms amyloid fibrils. PLoS ONE 13(1): e0191317. https://doi.org/ 10.1371/journal.pone.0191317

Based on the description in the passage amyloid fibrils would be classified as:

Amyloids are unbranched protein fibrils in which monomers form intermolecular β-sheets stabilized by numerous hydrogen bonds. This structure, called cross-β, gives amyloids unusual resistance to treatment with ionic detergents (such as SDS and sarkosyl). Initially, amyloids were described as the lethal pathogenic agents of incurable diseases called amyloidoses in humans. In recent decades, it has become clear that amyloids are not only involved in pathogenesis; they also play an essential role in a wide spectrum of biological processes.

A proteomic method called PSIA (Proteomic Screening and Identification of Amyloids) allows for the identification of candidates for amyloid-forming proteins in the complete proteomes of different organisms, was recently developed. Using PSIA scientists analyzed YghJ a secreted protein harboring an evolutionarily conserved zinc metalloprotease domain called M60 in E coli.

To analyze the properties of YghJM aggregation, scientists used a plasmid encoding YghJM C-terminally tagged with His6 and under the control of an IPTG-inducible T7 promoter. YghJM was then purified using column chromatography from E. coli and then transferred into non-denaturing conditions to allow spontaneous aggregation. These aggregates were then subjected to PSIA and the M60-like metalloprotease domain of YghJ was found to have a high amyloidogenic propensity: comprised of 3 individual amyloidogenic regions.

Adapted From:

Belousov MV, Bondarev SA, Kosolapova AO, Antonets KS, Sulatskaya AI, Sulatsky MI, et al. (2018) M60-like metalloprotease domain of the Escherichia coli YghJ protein forms amyloid fibrils. PLoS ONE 13(1): e0191317. https://doi.org/ 10.1371/journal.pone.0191317

Based on information in the passage amyloid fibrils would denature the most under conditions in which the:

Amyloids are unbranched protein fibrils in which monomers form intermolecular β-sheets stabilized by numerous hydrogen bonds. This structure, called cross-β, gives amyloids unusual resistance to treatment with ionic detergents (such as SDS and sarkosyl). Initially, amyloids were described as the lethal pathogenic agents of incurable diseases called amyloidoses in humans. In recent decades, it has become clear that amyloids are not only involved in pathogenesis; they also play an essential role in a wide spectrum of biological processes.

A proteomic method called PSIA (Proteomic Screening and Identification of Amyloids) allows for the identification of candidates for amyloid-forming proteins in the complete proteomes of different organisms, was recently developed. Using PSIA scientists analyzed YghJ a secreted protein harboring an evolutionarily conserved zinc metalloprotease domain called M60 in E coli.

To analyze the properties of YghJM aggregation, scientists used a plasmid encoding YghJM C-terminally tagged with His6 and under the control of an IPTG-inducible T7 promoter. YghJM was then purified using column chromatography from E. coli and then transferred into non-denaturing conditions to allow spontaneous aggregation. These aggregates were then subjected to PSIA and the M60-like metalloprotease domain of YghJ was found to have a high amyloidogenic propensity: comprised of 3 individual amyloidogenic regions.

Adapted From:

Belousov MV, Bondarev SA, Kosolapova AO, Antonets KS, Sulatskaya AI, Sulatsky MI, et al. (2018) M60-like metalloprotease domain of the Escherichia coli YghJ protein forms amyloid fibrils. PLoS ONE 13(1): e0191317. https://doi.org/ 10.1371/journal.pone.0191317

Samples from four patients who were suspected of having amyloidosis were run on both native and SDS PAGE gels. Based on the results which lane most likely contains a sample from a patient who has amyloidosis?