Chimeric elk/mouse prion proteins in transgenic mice

Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). Between codons 169 and 219, six residues distinguish ElkPrP from MoPrP: N169S, T173N, V183I, I202V, I214V and R219K. Using chimeric elk/mouse PrP constructs, we generated 12 Tg mouse lines and determined incubation times after intracerebral inoculation with the mouse-passaged RML scrapie or Elk1P CWD prions. Unexpectedly, one Tg mouse line expressing Elk3M(SNIVVK) exhibited incubation times of <70 days when inoculated with RML prions; a second line had incubation times of <90 days. In contrast, mice expressing full-length ElkPrP had incubation periods of >250 days for RML prions. Tg(Elk3M,SNIVVK) mice were less susceptible to CWD prions than Tg(ElkPrP) mice. Changing three C-terminal mouse residues (202, 214 and 219) to those of elk doubled the incubation time for mouse RML prions and rendered the mice resistant to Elk1P CWD prions. Mutating an additional two residues from mouse to elk at codons 169 and 173 increased the incubation times for mouse prions to >300 days, but made the mice susceptible to CWD prions. Our findings highlight the role of C-terminal residues in PrP that control the susceptibility and replication of prions.

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Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains

Biochemical Journal ◽

10.1042/bj2790787 ◽

1991 ◽

Vol 279 (3) ◽

pp. 787-792 ◽

Cited By ~ 22

Author(s):

D M Poole ◽

A J Durrant ◽

G P Hazlewood ◽

H J Gilbert

Keyword(s):

Catalytic Domain ◽

Binding Capacity ◽

Binding Domains ◽

Hybrid Proteins ◽

C Terminus ◽

N Terminus ◽

Fusion Enzymes ◽

Cellulose Binding

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

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Structure of APP-C991-99 and Implications for Role of Extra-Membrane Domains in Function and Oligomerization

10.1101/257469 ◽

2018 ◽

Author(s):

George A. Pantelopulos ◽

John E. Straub ◽

D. Thirumalai ◽

Yuji Sugita

Keyword(s):

Transmembrane Domain ◽

Chemical Shifts ◽

Membrane Surface ◽

Critical Role ◽

Amyloid Formation ◽

Cytoplasmic Proteins ◽

C Terminus ◽

N Terminus ◽

Thin Membranes

AbstractThe 99 amino acid C-terminal fragment of Amyloid Precursor Protein APP-C99 (C99) is cleaved by γ-secretase to form Aβ peptide, which plays a critical role in the etiology of Alzheimer’s Disease (AD). The structure of C99 consists of a single transmembrane domain flanked by intra and intercellular domains. While the structure of the transmembrane domain has been well characterized, little is known about the structure of the flanking domains and their role in C99 processing by γ-secretase. To gain insight into the structure of full-length C99, REMD simulations were performed for monomeric C99 in model membranes of varying thickness. We find equilibrium ensembles of C99 from simulation agree with experimentally-inferred residue insertion depths and protein backbone chemical shifts. In thin membranes, the transmembrane domain structure is correlated with extra-membrane structural states. Mean and variance of the transmembrane and G37G38 hinge angles are found to increase with thinning membrane. The N-terminus of C99 forms β-strands that may seed aggregation of Aβ on the membrane surface, promoting amyloid formation. The N-terminus, which forms α-helices that interact with the nicastrin domain of γ-secretase. The C-terminus of C99 becomes more α-helical as the membrane thickens, forming structures that may be suitable for binding by cytoplasmic proteins, while C-terminal residues essential to cytotoxic function become α-helical as the membrane thins. The heterogeneous but discrete extra-membrane domain states analyzed here open the path to new investigations of the role of C99 structure and membrane in amyloidogenesis.

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Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation

Journal of Cell Science ◽

10.1242/jcs.99.4.823 ◽

1991 ◽

Vol 99 (4) ◽

pp. 823-836

Author(s):

S.J. Atkinson ◽

M. Stewart

Keyword(s):

Escherichia Coli ◽

Ionic Strength ◽

Coiled Coil ◽

Periodic Variation ◽

Heptad Repeat ◽

C Terminus ◽

N Terminus ◽

Low Ionic Strength ◽

Myosin Rod

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.

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α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels

Molecular Biology of the Cell ◽

10.1091/mbc.e16-03-0163 ◽

2016 ◽

Vol 27 (16) ◽

pp. 2542-2553 ◽

Cited By ~ 17

Author(s):

Peiyao Li ◽

Yong Miao ◽

Adish Dani ◽

Monika Vig

Keyword(s):

Calcium Release ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Embryonic Fibroblasts ◽

Unique Role ◽

C Terminus ◽

Primary Mouse ◽

N Terminus ◽

Variable Stoichiometry

Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP–deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP–depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1.

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IN SILICO MODELING OF MONOMERIC DEXAMETHASONE-INDUCED RAS-RELATED PROTEIN 1 AND RAS HOMOLOG ENRICHED IN STRIATUM: ROLE OF N TERMINUS AND STRUCTURE‑FUNCTION RELATIONSHIP

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2018.v12i1.28486 ◽

2019 ◽

Vol 12 (1) ◽

pp. 84

Author(s):

Rashmi Verma ◽

Navin Kumar ◽

Ashish Thapliyal

Keyword(s):

3D Structure ◽

Three Dimensional ◽

Therapeutic Drug ◽

Related Protein ◽

Binding Domains ◽

Gtp Binding ◽

C Terminus ◽

N Terminus ◽

Function Relationship

Objective: Dexamethasone-induced Ras-related protein 1 (Dexras1) and Ras homolog enriched in striatum (RHES) are the two monomeric small G proteins that belong to Ras superfamily. These two proteins show 62% similarity. Both of these proteins are involved in signaling and modulation of several pathophysiological processes. They have unique GTP binding domain and a unique C and N terminus. C terminus is known to interact with several proteins; however, the role of its unique N terminus is still not known. The three-dimensional (3D) structure of these proteins is also not available in any of the databases yet. This present study approaches bioinformatics tools and servers to predict the 3D structure of these two proteins in silico.Methods: In this study, two bioinformatics servers were used, namely Swiss modeling server and Iterative Threading ASSEmbly Refinement (I-TASSER) server.Results: Both servers developed many alignment templates of Dexras1 and RHES. These alignments were used to develop 3D structure using Pymol. These models have different regions of proteins such as N terminus, GTP-binding domains, effector loop, C terminus, and the unique CAAX site. The models deduce that the N-terminals of both Dexras1 and RHES are unique regions that might possible be dangling out of the protein while it gets inserted into the membrane. We hypothesize that this unique N-terminal might have a distinct role in the modulation of N-type calcium channels.Conclusion: All the models generated show predicted 3D structure of Dexras1 and RHES protein. This study of structural prediction will be helpful in knowing the interaction of Dexras1 and RHES and a step forward to target these two proteins as a novel therapeutic drug.

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The Role of Mutant p63 in Female Fertility

International Journal of Molecular Sciences ◽

10.3390/ijms22168968 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8968

Author(s):

Yi Luan ◽

Pauline Xu ◽

Seok-Yeong Yu ◽

So-Youn Kim

Keyword(s):

Germ Cells ◽

Essential Role ◽

Primordial Germ Cells ◽

Genomic Integrity ◽

Biological Functions ◽

Ovarian Insufficiency ◽

P53 Family ◽

C Terminus ◽

N Terminus

The transcription factor p63, one of the p53 family members, plays an essential role in regulating maternal reproduction and genomic integrity as well as epidermal development. TP63 (human)/Trp63 (mouse) produces multiple isoforms: TAp63 and ΔNp63, which possess a different N-terminus depending on two different promoters, and p63a, p63b, p63g, p63δ, and p63ε as products of alternative splicing at the C-terminus. TAp63 expression turns on in the nuclei of primordial germ cells in females and is maintained mainly in the oocyte nuclei of immature follicles. It has been established that TAp63 is the genomic guardian in oocytes of the female ovaries and plays a central role in determining the oocyte fate upon oocyte damage. Lately, there is increasing evidence that TP63 mutations are connected with female infertility, including isolated premature ovarian insufficiency (POI) and syndromic POI. Here, we review the biological functions of p63 in females and discuss the consequences of p63 mutations, which result in infertility in human patients.

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Role of the N-terminus in human 4-hydroxyphenylpyruvate dioxygenase activity

The Journal of Biochemistry ◽

10.1093/jb/mvz092 ◽

2019 ◽

Vol 167 (3) ◽

pp. 315-322

Author(s):

An-Ning Feng ◽

Chih-Wei Huang ◽

Chi-Huei Lin ◽

Yung-Lung Chang ◽

Meng-Yuan Ni ◽

...

Keyword(s):

Active Site ◽

Catalytic Efficiency ◽

Dynamics Simulation ◽

Wild Type ◽

Catalytic Function ◽

C Terminus ◽

N Terminus ◽

Key Enzyme ◽

The Stability

Abstract 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism, catalysing the oxidation of 4-hydroxyphenylpyruvate to homogentisate. Genetic deficiency of this enzyme causes type III tyrosinaemia. The enzyme comprises two barrel-shaped domains formed by the N- and C-termini, with the active site located in the C-terminus. This study investigated the role of the N-terminus, located at the domain interface, in HPPD activity. We observed that the kcat/Km decreased ∼8-fold compared with wild type upon removal of the 12 N-terminal residues (ΔR13). Interestingly, the wild-type level of activity was retained in a mutant missing the 17 N-terminal residues, with a kcat/Km 11-fold higher than that of the ΔR13 mutant; however, the structural stability of this mutant was lower than that of wild type. A 2-fold decrease in catalytic efficiency was observed for the K10A and E12A mutants, indicating synergism between these residues in the enzyme catalytic function. A molecular dynamics simulation showed large RMS fluctuations in ΔR13 suggesting that conformational flexibility at the domain interface leads to lower activity in this mutant. These results demonstrate that the N-terminus maintains the stability of the domain interface to allow for catalysis at the active site of HPPD.

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Opposing actions of septins and Sticky on Anillin promote the transition from contractile to midbody ring

The Journal of Cell Biology ◽

10.1083/jcb.201305053 ◽

2013 ◽

Vol 203 (3) ◽

pp. 487-504 ◽

Cited By ~ 48

Author(s):

Nour El Amine ◽

Amel Kechad ◽

Silvana Jananji ◽

Gilles R.X. Hickson

Keyword(s):

Essential Role ◽

Time Lapse ◽

Maturation Process ◽

S2 Cells ◽

Contractile Ring ◽

Time Lapse Microscopy ◽

Drosophila Melanogaster S2 Cells ◽

C Terminus ◽

N Terminus

During cytokinesis, closure of the actomyosin contractile ring (CR) is coupled to the formation of a midbody ring (MR), through poorly understood mechanisms. Using time-lapse microscopy of Drosophila melanogaster S2 cells, we show that the transition from the CR to the MR proceeds via a previously uncharacterized maturation process that requires opposing mechanisms of removal and retention of the scaffold protein Anillin. The septin cytoskeleton acts on the C terminus of Anillin to locally trim away excess membrane from the late CR/nascent MR via internalization, extrusion, and shedding, whereas the citron kinase Sticky acts on the N terminus of Anillin to retain it at the mature MR. Simultaneous depletion of septins and Sticky not only disrupted MR formation but also caused earlier CR oscillations, uncovering redundant mechanisms of CR stability that can partly explain the essential role of Anillin in this process. Our findings highlight the relatedness of the CR and MR and suggest that membrane removal is coordinated with CR disassembly.

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Ndc80/Nuf2-like protein KKIP1 connects a stable kinetoplastid outer kinetochore complex to the inner kinetochore and responds to metaphase tension

10.1101/764829 ◽

2019 ◽

Cited By ~ 6

Author(s):

Lorenzo Brusini ◽

Simon D’Archivio ◽

Jennifer McDonald ◽

Bill Wickstead

Keyword(s):

Cell Biology ◽

Universal Function ◽

Model Organisms ◽

C Terminus ◽

N Terminus ◽

Core Components ◽

Moonlighting Proteins ◽

Nuclear Processes ◽

Sufficient Strength

AbstractKinetochores perform an essential role in eukaryotes, coupling chromosomes to the mitotic spindle. In model organisms they are composed of a centromere-proximal inner kinetochore and an outer kinetochore network that binds to microtubules. In spite of its universal function, the composition of kinetochores in extant eukaryotes can differ greatly, and understanding how these different systems evolved and now function are important questions in cell biology. In trypanosomes and other Kinetoplastida, the kinetochores are extremely divergent, with most components showing no detectable similarity to proteins in other systems. They may also be very different functionally, potentially binding to the spindle directly via the inner kinetochore protein KKT4. However, we do not know the extent of the trypanosome kinetochore and proteins interacting with a highly divergent Ndc80/Nuf2-like protein, KKIP1, suggest the existence of more centromere-distal complexes. Here we use quantitative proteomics from multiple start-points to define a stable 9-protein kinetoplastid outer kinetochore (KOK) complex. Two of these core components were recruited from other nuclear processes, exemplifying the role of moonlighting proteins in kinetochore evolution. The complex is physically and biochemically distinct from KKT proteins, but KKIP1 links the inner and outer sets, with its C-terminus very close to the centromere and N-terminus at the outer kinetochore. Moreover, trypanosome kinetochores exhibit intra-kinetochore movement during metaphase, primarily by elongation of KKIP1, consistent with pulling at the outer kinetochores. Together, these data suggest that the KOK complex, KKIP5 and N-terminus of KKIP1 likely constitute the extent of the trypanosome outer kinetochore and that this assembly binds to the spindle with sufficient strength to stretch the kinetochore.

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