scholarly journals Aggregation of prion protein with insertion mutations is proportional to the number of inserts

2007 ◽  
Vol 403 (2) ◽  
pp. 343-351 ◽  
Author(s):  
Shuiliang Yu ◽  
Shaoman Yin ◽  
Chaoyang Li ◽  
Poki Wong ◽  
Binggong Chang ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Diseases ◽  
Biochemical Properties ◽  
Mutant Protein ◽  
Insertion Mutant ◽  
Wild Type ◽  
Mutant Proteins ◽  
Severity Of The Disease ◽  
Α Helix ◽  
Insertion Mutations

Mutation in the prion gene, PRNP, accounts for approx. 10–15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrPC (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP8OR, and the other with five more octapeptide repeats, rPrP10OR. We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and α-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrPC. Accordingly, rPrP10OR is also more efficient in promoting the aggregation of rPrPC than rPrP8OR. These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.

scholarly journals Engineering of a Type III Rubisco from a Hyperthermophilic Archaeon in Order To Enhance Catalytic Performance in Mesophilic Host Cells

2007 ◽  
Vol 73 (19) ◽  
pp. 6254-6261 ◽  
Author(s):  
Shosuke Yoshida ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Rhodopseudomonas Palustris ◽  
Host Cells ◽  
Mutant Protein ◽  
Wild Type ◽  
Turnover Number ◽  
Bisphosphate Carboxylase ◽  
Mutant Proteins ◽  
Hyperthermophilic Archaeon ◽  
Type Iii ◽  
Α Helix

ABSTRACT The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a type III ribulose 1,5-bisphosphate carboxylase/oxygenase (RbcTk). It has previously been shown that RbcTk is capable of supporting photoautotrophic and photoheterotrophic growth in a mesophilic host cell, Rhodopseudomonas palustris Δ3, whose three native Rubisco genes had been disrupted. Here, we have examined the enzymatic properties of RbcTk at 25°C and have constructed mutant proteins in order to enhance its performance in mesophilic host cells. Initial sites for mutagenesis were selected by focusing on sequence differences in the loop 6 and α-helix 6 regions among RbcTk and the enzymes from spinach (mutant proteins SP1 to SP7), Galdieria partita (GP1 and GP2), and Rhodospirillum rubrum (RR1). Loop 6 of RbcTk is one residue longer than those found in the spinach and G. partita enzymes, and replacing RbcTk loop 6 with these regions led to dramatic decreases in activity. Six mutant enzymes retaining significant levels of Rubisco activity were selected, and their genes were introduced into R. palustris Δ3. Cells harboring mutant protein SP6 displayed a 31% increase in the specific growth rate under photoheterotrophic conditions compared to cells harboring wild-type RbcTk. SP6 corresponds to a complete substitution of the original α-helix 6 of RbcTk with that of the spinach enzyme. Compared to wild-type RbcTk, the purified SP6 mutant protein exhibited a 30% increase in turnover number (k cat) of the carboxylase activity and a 17% increase in the k cat/Km value. Based on these results, seven further mutant proteins were designed and examined. The results confirmed the importance of the length of loop 6 in RbcTk and also led to the identification of specific residue changes that resulted in an increase in the turnover number of RbcTk at ambient temperatures.

scholarly journals Complete Virion Assembly with Scaffolding Proteins Altered in the Ability To Perform a Critical Conformational Switch

2009 ◽  
Vol 83 (15) ◽  
pp. 7391-7396 ◽  
Author(s):  
James E. Cherwa ◽  
Bentley A. Fane
Keyword(s):  
Amino Acid ◽  
Scaffolding Proteins ◽  
Wild Type ◽  
Conformational Switch ◽  
Mutant Proteins ◽  
Virion Assembly ◽  
Glycine Residue ◽  
Atomic Structures ◽  
Dominant Lethal ◽  
Α Helix

ABSTRACT In the φX174 procapsid, 240 external scaffolding proteins form a nonquasiequivalent lattice. To achieve this arrangement, the four structurally unique subunits must undergo position-dependent conformational switches. One switch is mediated by glycine residue 61, which allows a 30° kink to form in α-helix 3 in two subunits, whereas the helix is straight in the other two subunits. No other amino acid should be able to produce a bend of this magnitude. Accordingly, all substitutions for G61 are nonviable but mutant proteins differ vis-à-vis recessive and dominant phenotypes. As previously reported, amino acid substitutions with side chains larger than valine confer dominant lethal phenotypes. Alone, these mutant proteins appear to have little or no biological activity but rather require the wild-type protein to interact with other structural proteins. Proteins with conservative substitutions for G61, serine and alanine, have now been characterized. Unlike the dominant lethal proteins, these proteins do not require wild-type subunits to interact with other viral proteins and cause assembly defects reminiscent of those conferred by the lethal dominant proteins in concert with wild-type subunits. Although atomic structures suggest that only a glycine residue can provide the proper torsion angle for assembly, mutants that can productively utilize the altered external scaffolding proteins were isolated, and the mutations were mapped to the coat and internal scaffolding proteins. Thus, the ability to isolate strains that could utilize the single mutant D protein species would not have been predicted from past structural analyses.

scholarly journals Aspartate 205 in the Catalytic Domain of Naphthalene Dioxygenase Is Essential for Activity

1999 ◽  
Vol 181 (6) ◽  
pp. 1831-1837 ◽  
Author(s):  
Rebecca E. Parales ◽  
Juanito V. Parales ◽  
David T. Gibson
Keyword(s):  
Electron Transfer ◽  
Oxygen Uptake ◽  
Catalytic Domain ◽  
Mutant Protein ◽  
Wild Type ◽  
Naphthalene Dioxygenase ◽  
Major Pathway ◽  
Α Subunit ◽  
Mutant Proteins ◽  
Mononuclear Iron

ABSTRACT The naphthalene dioxygenase enzyme system carries out the first step in the aerobic degradation of naphthalene byPseudomonas sp. strain NCIB 9816-4. The crystal structure of naphthalene dioxygenase (B. Kauppi, K. Lee, E. Carredano, R. E. Parales, D. T. Gibson, H. Eklund, and S. Ramaswamy, Structure 6:571–586, 1998) indicates that aspartate 205 may provide the most direct route of electron transfer between the Rieske [2Fe-2S] center of one α subunit and mononuclear iron in the adjacent α subunit. In this study, we constructed four site-directed mutations that changed aspartate 205 to alanine, glutamate, asparagine, or glutamine to test whether this residue is essential for naphthalene dioxygenase activity. The mutant proteins were very inefficient in oxidizing naphthalene tocis-naphthalene dihydrodiol, and oxygen uptake in the presence of naphthalene was below detectable levels. The purified mutant protein with glutamine in place of aspartate 205 had identical spectral properties to wild-type naphthalene dioxygenase and was reduced by NADH in the presence of catalytic amounts of ferredoxinNAP and reductaseNAP. Benzene, an effective uncoupler of oxygen consumption in purified naphthalene dioxygenase, did not elicit oxygen uptake by the mutant protein. These results indicate that electron transfer from NADH to the Rieske center in the mutant oxygenase is intact, a finding consistent with the proposal that aspartate 205 is a necessary residue in the major pathway of electron transfer to mononuclear iron at the active site.

Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity

1991 ◽  
Vol 11 (7) ◽  
pp. 3537-3544 ◽  
Author(s):  
M Ziman ◽  
J M O'Brien ◽  
L A Ouellette ◽  
W R Church ◽  
D I Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Mutational Analysis ◽  
Mutant Gene ◽  
Biochemical Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Dominant Lethal ◽  
Gtp Binding ◽  
Multiple Buds

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

scholarly journals Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity.

1991 ◽  
Vol 11 (7) ◽  
pp. 3537-3544 ◽  
Author(s):  
M Ziman ◽  
J M O'Brien ◽  
L A Ouellette ◽  
W R Church ◽  
D I Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Mutational Analysis ◽  
Mutant Gene ◽  
Biochemical Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Dominant Lethal ◽  
Gtp Binding ◽  
Multiple Buds

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

scholarly journals Prion Protein Devoid of the Octapeptide Repeat Region Delays Bovine Spongiform Encephalopathy Pathogenesis in Mice

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Hideyuki Hara ◽  
Hironori Miyata ◽  
Nandita Rani Das ◽  
Junji Chida ◽  
Tatenobu Yoshimochi ◽  
...  
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Prion Protein ◽  
Prion Diseases ◽  
Spongiform Encephalopathy ◽  
Wild Type ◽  
Jakob Disease ◽  
Function Relationship ◽  
Different Strains ◽  
Conformational Conversion

ABSTRACTConformational conversion of the cellular isoform of prion protein, PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals. We previously reported that the octapeptide repeat (OR) region could be dispensable for converting PrPCinto PrPScafter infection with RML prions. We demonstrated that mice transgenically expressing mouse PrP with deletion of the OR region on the PrP knockout background, designated Tg(PrPΔOR)/Prnp0/0mice, did not show reduced susceptibility to RML scrapie prions, with abundant accumulation of PrPScΔOR in their brains. We show here that Tg(PrPΔOR)/Prnp0/0mice were highly resistant to BSE prions, developing the disease with markedly elongated incubation times after infection with BSE prions. The conversion of PrPΔOR into PrPScΔOR was markedly delayed in their brains. These results suggest that the OR region may have a crucial role in the conversion of PrPCinto PrPScafter infection with BSE prions. However, Tg(PrPΔOR)/Prnp0/0mice remained susceptible to RML and 22L scrapie prions, developing the disease without elongated incubation times after infection with RML and 22L prions. PrPScΔOR accumulated only slightly less in the brains of RML- or 22L-infected Tg(PrPΔOR)/Prnp0/0mice than PrPScin control wild-type mice. Taken together, these results indicate that the OR region of PrPCcould play a differential role in the pathogenesis of BSE prions and RML or 22L scrapie prions.IMPORTANCEStructure-function relationship studies of PrPCconformational conversion into PrPScare worthwhile to understand the mechanism of the conversion of PrPCinto PrPSc. We show here that, by inoculating Tg(PrPΔOR)/Prnp0/0mice with the three different strains of RML, 22L, and BSE prions, the OR region could play a differential role in the conversion of PrPCinto PrPScafter infection with RML or 22L scrapie prions and BSE prions. PrPΔOR was efficiently converted into PrPScΔOR after infection with RML and 22L prions. However, the conversion of PrPΔOR into PrPScΔOR was markedly delayed after infection with BSE prions. Further investigation into the role of the OR region in the conversion of PrPCinto PrPScafter infection with BSE prions might be helpful for understanding the pathogenesis of BSE prions.

The neuropathological phenotype in transgenic mice expressing different prion protein constructs

1994 ◽  
Vol 343 (1306) ◽  
pp. 415-423 ◽  
Keyword(s):  
Prion Protein ◽  
Prion Diseases ◽  
Amyloid Plaques ◽  
Pathogenic Factor ◽  
Wild Type ◽  
Vacuolar Degeneration ◽  
Neuron Populations ◽  
Age Related ◽  
Human Prion Protein ◽  
The Brain

Neuropathologic examination of transgenic (Tg) mice which express different prion protein (PrP) constructs is essential because spongiform (vacuolar) degeneration of neurons, the distribution of PrP Sc and whether PrP amyloid plaques form are the phenotypes of prion diseases. In Tg models of experimental scrapie, it was found that all of the parameters that define prion isolates (‘strains’) can be manipulated by changing the structure of PrP. In those studies, further evidence that PrP Sc causes scrapie neuropathology and determines scrapie incubation time was obtained. In addition, the distribution of PrP Sc in the brain was unique for each prion isolate. The implications of these findings are first, that prion isolates target different neuron populations for synthesis of nascent pathogenic PrP Sc and, secondly, that prion isolate diversity is determined by neurons. In Tg mice which express mutated PrP mimicking human prion protein genemutations linked to familial prion diseases, the neuropathological changes have been faithfully reproduced. A new age-related, neuromascular disorder has also been identified in uninfected Tg mice which overexpress wild-type PrP C . All of the findings with different PrP constructs plus the absence of scrapie pathology in PrP null mice are the strongest argument that the prion protein is the main etiologic and pathogenic factor of prion disorders.

scholarly journals New and distinct chronic wasting disease strains associated with cervid polymorphism at codon 116 of the Prnp gene

2021 ◽  
Vol 17 (7) ◽  
pp. e1009795
Author(s):  
Samia Hannaoui ◽  
Elizabeth Triscott ◽  
Camilo Duque Velásquez ◽  
Sheng Chun Chang ◽  
Maria Immaculata Arifin ◽  
...  
Keyword(s):  
Prion Protein ◽  
Chronic Wasting Disease ◽  
Prnp Gene ◽  
Biochemical Properties ◽  
Cellular Prion Protein ◽  
Proteinase K ◽  
Wild Type ◽  
Wasting Disease ◽  
Prion Strains ◽  
The Impact

Chronic wasting disease (CWD) is a prion disease affecting cervids. Polymorphisms in the prion protein gene can result in extended survival of CWD-infected animals. However, the impact of polymorphisms on cellular prion protein (PrPC) and prion properties is less understood. Previously, we characterized the effects of a polymorphism at codon 116 (A>G) of the white-tailed deer (WTD) prion protein and determined that it destabilizes PrPC structure. Comparing CWD isolates from WTD expressing homozygous wild-type (116AA) or heterozygous (116AG) PrP, we found that 116AG-prions were conformationally less stable, more sensitive to proteases, with lower seeding activity in cell-free conversion and reduced infectivity. Here, we aimed to understand CWD strain emergence and adaptation. We show that the WTD-116AG isolate contains two different prion strains, distinguished by their host range, biochemical properties, and pathogenesis from WTD-116AA prions (Wisc-1). Serial passages of WTD-116AG prions in tg(CerPrP)1536+/+ mice overexpressing wild-type deer-PrPC revealed two populations of mice with short and long incubation periods, respectively, and remarkably prolonged clinical phase upon inoculation with WTD-116AG prions. Inoculation of serially diluted brain homogenates confirmed the presence of two strains in the 116AG isolate with distinct pathology in the brain. Interestingly, deglycosylation revealed proteinase K-resistant fragments with different electrophoretic mobility in both tg(CerPrP)1536+/+ mice and Syrian golden hamsters infected with WTD-116AG. Infection of tg60 mice expressing deer S96-PrP with 116AG, but not Wisc-1 prions induced clinical disease. On the contrary, bank voles resisted 116AG prions, but not Wisc-1 infection. Our data indicate that two strains co-existed in the WTD-116AG isolate, expanding the variety of CWD prion strains. We argue that the 116AG isolate does not contain Wisc-1 prions, indicating that the presence of 116G-PrPC diverted 116A-PrPC from adopting a Wisc-1 structure. This can have important implications for their possible distinct capacities to cross species barriers into both cervids and non-cervids.

Biophysical characterization of oligomerization and fibrillization of the G131V pathogenic mutant of human prion protein

2019 ◽  
Author(s):  
Meilan Zhang ◽  
Haoran Zhang ◽  
Hongwei Yao ◽  
Chenyun Guo ◽  
Donghai Lin
Keyword(s):  
Prion Protein ◽  
Tertiary Structure ◽  
Prion Proteins ◽  
Prion Diseases ◽  
Guanidine Hydrochloride ◽  
Point Mutations ◽  
Cellular Prion Protein ◽  
Human Prion Protein ◽  
Α Helix ◽  
Conformational Conversion

Abstract The pathogenesis of fatal neurodegenerative prion diseases is closely associated with the conversion of α-helix-rich cellular prion protein into β-sheet-rich scrapie form. Pathogenic point mutations of prion proteins usually promote the conformational conversion and trigger inherited prion diseases. The G131V mutation of human prion protein (HuPrP) was identified to be involved in Gerstmann–Sträussler–Scheinker syndrome. Few studies have been carried out to address the pathogenesis of the G131V mutant. Here, we addressed the effects of the G131V mutation on oligomerization and fibrillization of the full-length HuPrP(23–231) and truncated HuPrP(91–231) proteins. The G131V mutation promotes the oligomerization but alleviates the fibrillization of HuPrP, implying that the oligomerization might play a crucial role in the pathogenic mechanisms of the G131V mutant. Moreover, the flexible N-terminal fragment in either the wild-type or the G131V mutant HuPrP increases the oligomerization tendencies but decreases the fibrillization tendencies. Furthermore, this mutation significantly alters the tertiary structure of human PrPC and might distinctly change the conformational conversion tendency. Interestingly, both guanidine hydrochloride denaturation and thermal denaturation experiments showed that the G131V mutation does not significantly change the thermodynamic stabilities of the HuPrP proteins. This work may be of benefit to a mechanistic understanding of the conformational conversion of prion proteins and also provide clues for the prevention and treatment of prion diseases.

scholarly journals Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport.

1995 ◽  
Vol 130 (5) ◽  
pp. 1051-1061 ◽  
Author(s):  
S Jones ◽  
R J Litt ◽  
C J Richardson ◽  
N Segev
Keyword(s):  
Essential Role ◽  
Protein Transport ◽  
Vesicular Transport ◽  
Mutant Protein ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Mutant Proteins ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor

Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein, and are potent inhibitors of ER to Golgi transport in vitro at the vesicle targeting step. The inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide exchange activity and protein transport in vitro can be relieved by XTP, indicating that it is the nucleotide-free form of the mutant protein that is inhibitory. These results suggest that the dominant mutant proteins inhibit protein transport by sequestering the exchange factor from the wild type Ypt1 protein, and that this factor has an essential role in vesicular transport.

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