scholarly journals A systematic mutational analysis identifies a 5‐residue proline tag that enhances the in vivo immunogenicity of a non‐immunogenic model protein

FEBS Open Bio ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1947-1956
Author(s):  
Nafsoon Rahman ◽  
Mohammad Monirul Islam ◽  
Md. Golam Kibria ◽  
Satoru Unzai ◽  
Yutaka Kuroda
Keyword(s):  
Mutational Analysis ◽  
Model Protein

scholarly journals A systematic mutational analysis identifies a 5-residue proline tag that enhances the in vivo immunogenicity of a non-immunogenic model protein 240 folds

2020 ◽  
Author(s):  
Nafsoon Rahman ◽  
Mohammad Monirul Islam ◽  
Md Golam Kibria ◽  
Satoru Unzai ◽  
Yutaka Kuroda
Keyword(s):  
Mutational Analysis ◽  
Peptide Vaccines ◽  
Tertiary Structures ◽  
Small Proteins ◽  
C Terminus ◽  
Pancreatic Trypsin Inhibitor ◽  
Model Protein ◽  
Soluble Aggregates ◽  
Igg Level

AbstractSmall proteins are generally non-immunogenic, which can be a major hurdle in developing protein and peptide vaccines or producing antibodies for biopharmaceutical usage. For improving a protein’s immunogenicity, we previously proposed to use short Solubility Controlling Peptide (SCP) tags that oligomerize proteins into soluble aggregates. Here, we systematically analyzed the effect of SCP-tags that do not induce oligomerization on the immunogenicity of a small, non-immunogenic, model protein, Bovine Pancreatic Trypsin Inhibitor (BPTI-19A; 6 kDa). We assessed the effect of the following ten SCP-tags: Six tags made of five consecutive Arg, Lys, His, Asp, Asn, Pro; one made of seven Pro; two tags made of consecutive Arg-lle and Asn-Ile, all attached at the C-terminus of BPTI-19A; and a 5-proline tag attached at the N-terminus. Circular dichroism, fluorescence, dynamic light scattering measurements, and analytical ultra-centrifugation indicated that the addition of the SCP-tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. On the other hand, the C-terminus 5-proline (C5P) tag unexpectedly increased the immunogenicity (IgG level) of BPTI-19A by up to 240 fold as assessed by ELISA. Additionally, the 5-arginine tag (C5R) increased the titer by up to 73 fold. The titer increase lasted for several weeks, and the effect was cumulative to that of the Freund’s adjuvant, which is commonly used to boost a protein’s immunogenicity. Altogether, SCP-tags that do not oligomerize proteins substantially increased the immunogenicity of a non-immunogenic protein, suggesting that the 5-proline and the 5-arginine SCP-tags may provide a novel tool for facilitating the production of antibodies or improving the effectiveness of protein-based vaccines.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catalyzed by the Salmonella Hin Recombinase

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1649-1663
Author(s):  
Oliver Z Nanassy ◽  
Kelly T Hughes
Keyword(s):  
Amino Acids ◽  
Biochemical Analysis ◽  
Mutational Analysis ◽  
Recombination Reaction ◽  
Recombination Site ◽  
Dna Inversion ◽  
Hin Recombinase ◽  
One Step ◽  
A Site

Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

scholarly journals Characterization of the SARS-CoV-2 coronavirus X4-like accessory protein

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Olanrewaju Ayodeji Durojaye ◽  
Nkwachukwu Oziamara Okoro ◽  
Arome Solomon Odiba
Keyword(s):  
Rapid Development ◽  
Protein A ◽  
The Novel ◽  
Quality Model ◽  
A Value ◽  
Model Protein ◽  
Novel Coronavirus ◽  
Global Threat

Abstract Background The novel coronavirus SARS-CoV-2 is currently a global threat to health and economies. Therapeutics and vaccines are in rapid development; however, none of these therapeutics are considered as absolute cure, and the potential to mutate makes it necessary to find therapeutics that target a highly conserved regions of the viral structure. Results In this study, we characterized an essential but poorly understood coronavirus accessory X4 protein, a core and stable component of the SARS-CoV family. Sequence analysis shows a conserved ~ 90% identity between the SARS-CoV-2 and previously characterized X4 protein in the database. QMEAN Z score of the model protein shows a value of around 0.5, within the acceptable range 0–1. A MolProbity score of 2.96 was obtained for the model protein and indicates a good quality model. The model has Ramachandran values of φ = − 57o and ψ = − 47o for α-helices and values of φ = − 130o and ψ = + 140o for twisted sheets. Conclusions The protein data obtained from this study provides robust information for further in vitro and in vivo experiment, targeted at devising therapeutics against the virus. Phylogenetic analysis further supports previous evidence that the SARS-CoV-2 is positioned with the SL-CoVZC45, BtRs-BetaCoV/YN2018B and the RS4231 Bat SARS-like corona viruses.

scholarly journals dDYRK2: a novel dual-specificity tyrosine-phosphorylation-regulated kinase in Drosophila

2003 ◽  
Vol 374 (2) ◽  
pp. 381-391 ◽  
Author(s):  
Pamela A. LOCHHEAD ◽  
Gary SIBBET ◽  
Ross KINSTRIE ◽  
Tava CLEGHON ◽  
Margie RYLATT ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Protein Kinases ◽  
Mutational Analysis ◽  
Kinase Domain ◽  
Activation Loop ◽  
Embryonic Neurogenesis ◽  
Founding Family ◽  
Dual Specificity ◽  
Eukaryotic Organisms

Dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are an emerging family of protein kinases that have been identified in all eukaryotic organisms examined to date. DYRK family members are involved in regulating key developmental and cellular processes such as neurogenesis, cell proliferation, cytokinesis and cellular differentiation. Two distinct subgroups exist, nuclear and cytosolic. In Drosophila, the founding family member minibrain, whose human orthologue maps to the Down syndrome critical region, belongs to the nuclear subclass and affects post-embryonic neurogenesis. In the present paper, we report the isolation of dDYRK2, a cytosolic DYRK and the putative product of the smell-impaired smi35A gene. This is the second such kinase described in Drosophila, but the first to be characterized at the molecular and biochemical level. dDYRK2 is an 81 kDa dual-specificity kinase that autophosphorylates on tyrosine and serine/threonine residues, but appears to phosphorylate exogenous substrates only on serine/threonine residues. It contains a YXY motif in the activation loop of the kinase domain in the same location as the TXY motif in mitogenactivated protein kinases. dDYRK2 is tyrosine-phosphorylated in vivo, and mutational analysis reveals that the activation loop tyrosines are phosphorylated and are essential for kinase activity. Finally, dDYRK2 is active at all stages of fly development, with elevated levels observed during embryogenesis and pupation.

scholarly journals Dissection of Swa2p/Auxilin Domain Requirements for Cochaperoning Hsp70 Clathrin-uncoating Activity In Vivo

2006 ◽  
Vol 17 (7) ◽  
pp. 3281-3290 ◽  
Author(s):  
Jing Xiao ◽  
Leslie S. Kim ◽  
Todd R. Graham
Keyword(s):  
Atpase Activity ◽  
Point Mutation ◽  
Mutational Analysis ◽  
Weak Interactions ◽  
Tetratricopeptide Repeat ◽  
Uba Domain ◽  
J Domain ◽  
Tpr Domain

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.

Application of middle-down approach in quantitation and catabolite identification of protein by LC–high-resolution mass spectrometry

Bioanalysis ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 465-479
Author(s):  
Lijuan Kang ◽  
Shengsheng Xu ◽  
Yongle Pang ◽  
Tom Kirchner ◽  
Yue-mei Zhang ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Resolving Power ◽  
Detection Sensitivity ◽  
Intact Protein ◽  
Top Down ◽  
Assay Performance ◽  
Model Protein ◽  
Bioanalytical Assay ◽  
Signal Improvement

Aim: To further enhance the detection sensitivity and increase resolving power of top-down intact protein bioanalysis, middle-down approach was explored. Materials & methods: An monoclonal antibody (mAb) was used as a model protein to evaluate quantitative bioanalytical assay performance and a disulfide linked dimer protein was investigated for its pharmacokinetics properties and catabolism in vivo by middle-down approach. Results & Conclusion: For quantitation of the mAb, different subunits generated by middle-down approach provided different level of signal improvement in biological samples with Lc and half Fc giving five-times better sensitivity than intact mAb. For the dimer protein, middle-down analysis by reduction enabled effective differentiation of the unchanged protein and its oxidized form, and clearly elucidated their respective proteolytic catabolites.

scholarly journals The Middle Domain of Hsp90 Acts as a Discriminator between Different Types of Client Proteins

2006 ◽  
Vol 26 (22) ◽  
pp. 8385-8395 ◽  
Author(s):  
Patricija Hawle ◽  
Martin Siepmann ◽  
Anja Harst ◽  
Marco Siderius ◽  
H. Peter Reusch ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Fold Increase ◽  
Cellular Protein ◽  
Hydrolysis Rate ◽  
Cellular Activity ◽  
Protein Levels ◽  
Middle Domain ◽  
Client Proteins

ABSTRACT The mechanism of client protein activation by Hsp90 is enigmatic, and it is uncertain whether Hsp90 employs a common route for all proteins. Using a mutational analysis approach, we investigated the activation of two types of client proteins, glucocorticoid receptor (GR) and the kinase v-Src by the middle domain of Hsp90 (Hsp90M) in vivo. Remarkably, the overall cellular activity of v-Src was highly elevated in a W300A mutant yeast strain due to a 10-fold increase in cellular protein levels of the kinase. In contrast, the cellular activity of GR remained almost unaffected by the W300A mutation but was dramatically sensitive to S485Y and T525I exchanges. In addition, we show that mutations S485Y and T525I in Hsp90M reduce the ATP hydrolysis rate, suggesting that Hsp90 ATPase is more tightly regulated than assumed previously. Therefore, the activation of GR and v-Src has various demands on Hsp90 biochemistry and is dependent on separate functional regions of Hsp90M. Thus, Hsp90M seems to discriminate between different substrate types and to adjust the molecular chaperone for proper substrate activation.

Sign in / Sign up

Export Citation Format

Share Document