The spliceosomal proteins PPIH and PRPF4 exhibit bi-partite binding

Pre-mRNA splicing is a dynamic, multistep process that is catalyzed by the RNA (ribonucleic acid)–protein complex called the spliceosome. The spliceosome contains a core set of RNAs and proteins that are conserved in all organisms that perform splicing. In higher organisms, peptidyl-prolyl isomerase H (PPIH) directly interacts with the core protein pre-mRNA processing factor 4 (PRPF4) and both integrate into the pre-catalytic spliceosome as part of the tri-snRNP (small nuclear RNA–protein complex) subcomplex. As a first step to understand the protein interactions that dictate PPIH and PRPF4 function, we expressed and purified soluble forms of each protein and formed a complex between them. We found two sites of interaction between PPIH and the N-terminus of PRPF4, an unexpected result. The N-terminus of PRPF4 is an intrinsically disordered region and does not adopt secondary structure in the presence of PPIH. In the absence of an atomic resolution structure, we used mutational analysis to identify point mutations that uncouple these two binding sites and find that mutations in both sites are necessary to break up the complex. A discussion of how this bipartite interaction between PPIH and PRPF4 may modulate spliceosomal function is included.

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Identification of Functionally Important Amino Acids of Ribosomal Protein L3 by Saturation Mutagenesis

Molecular and Cellular Biology ◽

10.1128/mcb.25.24.10863-10874.2005 ◽

2005 ◽

Vol 25 (24) ◽

pp. 10863-10874 ◽

Cited By ~ 39

Author(s):

Arturas Meskauskas ◽

Alexey N. Petrov ◽

Jonathan D. Dinman

Keyword(s):

Ribosomal Protein ◽

Protein Interactions ◽

Ribosomal Proteins ◽

Large Scale ◽

Mutational Analysis ◽

Core Protein ◽

Large Subunit ◽

Saturation Mutagenesis ◽

Functional Regions ◽

Ribosomal Protein L3

ABSTRACT There is accumulating evidence that many ribosomal proteins are involved in shaping rRNA into their functionally correct conformations through RNA-protein interactions. Moreover, although rRNA seems to play the central role in all aspects of ribosome function, ribosomal proteins may be involved in facilitating communication between different functional regions in ribosome, as well as between the ribosome and cellular factors. In an effort to more fully understand how ribosomal proteins may influence ribosome function, we undertook large-scale mutational analysis of ribosomal protein L3, a core protein of the large subunit that has been implicated in numerous ribosome-associated functions in the past. A total of 98 different rpl3 alleles were genetically characterized with regard to their effects on killer virus maintenance, programmed −1 ribosomal frameshifting, resistance/hypersensitivity to the translational inhibitor anisomycin and, in specific cases, the ability to enhance translation of a reporter mRNA lacking the 5′ 7mGppp cap structure and 3′ poly(A) tail. Biochemical studies reveal a correlation between an increased affinity for aminoacyl-tRNA and the extent of anisomycin resistance and a decreased peptidyltransferase activity and increased frameshifting efficiency. Immunoblot analyses reveal that the superkiller phenotype is not due to a defect in the ability of ribosomes to recruit the Ski-complex, suggesting that the defect lies in a reduced ability of mutant ribosomes to distinguish between cap+/poly(A)+ and cap−/poly(A)− mRNAs. The results of these analyses are discussed with regard to how protein-rRNA interactions may affect ribosome function.

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Faculty Opinions recommendation of Analysis of protein dynamics within the septate junction reveals a highly stable core protein complex that does not include the basolateral polarity protein Discs large.

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

10.3410/f.717955669.793460673 ◽

2012 ◽

Author(s):

Yang Hong

Keyword(s):

Protein Dynamics ◽

Protein Complex ◽

Core Protein ◽

Septate Junction ◽

Discs Large ◽

Stable Core

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SUMO's Intrinsically Disordered N-Terminus is an Intramolecular Inhibitor of SUMO - SIM Interactions

SSRN Electronic Journal ◽

10.2139/ssrn.3554087 ◽

2020 ◽

Author(s):

Sebastian M. Richter ◽

Fan Jin ◽

Eric Maurer ◽

Annette Flotho ◽

Frauke Gräter ◽

...

Keyword(s):

Intrinsically Disordered ◽

N Terminus

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Mathematical and Computational Techniques for Drug Discovery: Promises and Developments

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666190208164005 ◽

2019 ◽

Vol 18 (32) ◽

pp. 2774-2799 ◽

Cited By ~ 8

Author(s):

Krishnan Balasubramanian

Keyword(s):

Drug Discovery ◽

Protein Interactions ◽

Quantum Chemical ◽

Intrinsically Disordered Proteins ◽

Hepatitis Virus ◽

Disordered Proteins ◽

Computational Techniques ◽

Large Set ◽

Structural Protein ◽

Intrinsically Disordered

We review various mathematical and computational techniques for drug discovery exemplifying some recent works pertinent to group theory of nested structures of relevance to phylogeny, topological, computational and combinatorial methods for drug discovery for multiple viral infections. We have reviewed techniques from topology, combinatorics, graph theory and knot theory that facilitate topological and mathematical characterizations of protein-protein interactions, molecular-target interactions, proteomics, genomics and statistical data reduction procedures for a large set of starting chemicals in drug discovery. We have provided an overview of group theoretical techniques pertinent to phylogeny, protein dynamics especially in intrinsically disordered proteins, DNA base permutations and related algorithms. We consider computational techniques derived from high level quantum chemical computations such as QM/MM ONIOM methods, quantum chemical optimization of geometries complexes, and molecular dynamics methods for providing insights into protein-drug interactions. We have considered complexes pertinent to Hepatitis Virus C non-structural protein 5B polymerase receptor binding of C5-Arylidebne rhodanines, complexes of synthetic potential vaccine molecules with dengue virus (DENV) and HIV-1 virus as examples of various simulation studies that exemplify the utility of computational tools. It is demonstrated that these combinatorial and computational techniques in conjunction with experiments can provide promising new insights into drug discovery. These techniques also demonstrate the need to consider a new multiple site or allosteric binding approach to drug discovery, as these studies reveal the existence of multiple binding sites.

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MUTATIONAL ANALYSIS OF THE REGION SURROUNDING THE 93D HEAT SHOCK LOCUS OF DROSOPHILA MELANOGASTER

Genetics ◽

10.1093/genetics/106.2.249 ◽

1984 ◽

Vol 106 (2) ◽

pp. 249-265

Author(s):

Jym Mohler ◽

Mary Lou Pardue

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Mutational Analysis ◽

Point Mutations ◽

Γ Irradiation ◽

Lethal Mutations ◽

Complementation Groups ◽

In Trans ◽

Shock Locus ◽

Shock Proteins

ABSTRACT The region containing subdivisions 93C, 93D and 93E on chromosome 3 of Drosophila melanogaster has been screened for visible and lethal mutations. Treatment with three mutagens, γ irradiation, ethyl methanesulfonate and diepoxybutane, has produced mutations that fall into 20 complementation groups, including the previously identified ebony locus. No point mutations affecting the heat shock locus in 93D were detected; however, a pair of deficiencies that overlap in the region of this locus was isolated. Flies heterozygous in trans for this pair of deficiencies are capable of producing all of the major heat shock puffs (except 93D) and the major heat shock proteins. In addition, these flies show recovery of normal protein synthesis following a heat shock.

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

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

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.

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TBP and SNAP50 transcription factors bind specifically to the Pr77 promoter sequence from trypanosomatid non-LTR retrotransposons

Parasites & Vectors ◽

10.1186/s13071-021-04803-5 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Francisco Macías ◽

Raquel Afonso-Lehmann ◽

Patricia E. Carreira ◽

M. Carmen Thomas

Keyword(s):

Transcription Factors ◽

Protein Interactions ◽

Dna Sequences ◽

Direct Interaction ◽

Promoter Sequence ◽

Nuclear Proteins ◽

Hill Coefficient ◽

Small Nuclear Rna ◽

Mobile Dna ◽

Mobility Shift

Abstract Background Trypanosomatid genomes are colonized by active and inactive mobile DNA elements, such as LINE, SINE-like, SIDER and DIRE retrotransposons. These elements all share a 77-nucleotide-long sequence at their 5′ ends, known as Pr77, which activates transcription, thereby generating abundant unspliced and translatable transcripts. However, transcription factors that mediates this process have still not been reported. Methods TATA-binding protein (TBP) and small nuclear RNA-activating protein 50 kDa (SNAP50) recombinant proteins and specific antibodies raised against them were generated. Protein capture assay, electrophoretic mobility-shift assays (EMSA) and EMSA competition assays carried out using these proteins and nuclear proteins of the parasite together to specific DNA sequences used as probes allowed detecting direct interaction of these transcription factors to Pr77 sequence. Results This study identified TBP and SNAP50 as part of the DNA-protein complex formed by the Pr77 promoter sequence and nuclear proteins of Trypanosoma cruzi. TBP establishes direct and specific contact with the Pr77 sequence, where the DPE and DPE downstream regions are docking sites with preferential binding. TBP binds cooperatively (Hill coefficient = 1.67) to Pr77 and to both strands of the Pr77 sequence, while the conformation of this highly structured sequence is not involved in TBP binding. Direct binding of SNAP50 to the Pr77 sequence is weak and may be mediated by protein–protein interactions through other trypanosomatid nuclear proteins. Conclusions Identification of the transcription factors that mediate Pr77 transcription may help to elucidate how these retrotransposons are mobilized within the trypanosomatid genomes and their roles in gene regulation processes in this human parasite. Graphic abstract

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Specificity and VH sequence of two monoclonal antibodies against the N-terminus of dystrophin

Biochemical Journal ◽

10.1042/bj3090355 ◽

1995 ◽

Vol 309 (1) ◽

pp. 355-359 ◽

Cited By ~ 6

Author(s):

G E Morris ◽

C Nguyen ◽

Nguyen thi Man

Keyword(s):

Monoclonal Antibodies ◽

Point Mutations ◽

Hypervariable Region ◽

Variable Region ◽

Binding Studies ◽

Cdna Sequences ◽

N Terminus ◽

Random Library ◽

Blast Cells

We have used a random library of 15-mer peptides expressed on phage to show that two monoclonal antibodies (mAbs) require only the first three amino acids of dystrophin (Leu-Trp-Trp) for binding. Since the mAbs recognize dystrophin in frozen muscle sections, the results suggest that this hydrophobic N-terminus of dystrophin is accessible to antibody in situ. Quantitative binding studies suggested minor differences in specificity between the two mAbs, so the Ig heavy-chain variable region (VH) sequences of the two hybridomas were determined by RT-PCR and cDNA sequencing. After elimination of PCR errors, the two cDNA sequences were found to be identical except for five somatic mutations which resulted in three amino acid changes in the second hypervariable region (CDR2). The results suggest that the two hybridomas originated from the same lymphocyte clone in a germinal centre of the spleen, but underwent different point mutations and subtype switches during clonal expansion to form blast cells.

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Determinants That Control the Specific Interactions between TAB1 and p38α

Molecular and Cellular Biology ◽

10.1128/mcb.26.10.3824-3834.2006 ◽

2006 ◽

Vol 26 (10) ◽

pp. 3824-3834 ◽

Cited By ~ 24

Author(s):

Huamin Zhou ◽

Min Zheng ◽

Jianming Chen ◽

Changchuan Xie ◽

Anand R. Kolatkar ◽

...

Keyword(s):

Protein Kinase ◽

Conformational Changes ◽

Transforming Growth Factor ◽

Mutational Analysis ◽

Specific Binding ◽

Point Mutations ◽

Transforming Growth Factor Β ◽

Mitogen Activated Protein Kinase ◽

Docking Site ◽

C Terminus

ABSTRACT Previous studies have revealed that transforming growth factor-β-activated protein kinase 1 (TAB1) interacts with p38α and induces p38α autophosphorylation. Here, we examine the sequence requirements in TAB1 and p38α that drive their interaction. Deletion and point mutations in TAB1 reveal that a proline residue in the C terminus of TAB1 (Pro412) is necessary for its interaction with p38α. Furthermore, a cryptic D-domain-like docking site was identified adjacent to the N terminus of Pro412, putting Pro412 in the φB+3 position of the docking site. Through mutational analysis, we found that the previously identified hydrophobic docking groove in p38α is involved in this interaction, whereas the CD domain and ED domain are not. Furthermore, chimeric analysis with p38β (which does not bind to TAB1) revealed a previously unidentified locus of p38α comprising Thr218 and Ile275 that is essential for specific binding of p38α to TAB1. Converting either of these residues to the corresponding amino acid of p38β abolishes p38α interaction with TAB1. These p38α mutants still can be fully activated by p38α upstream activating kinase mitogen-activated protein kinase kinase 6, but their basal activity and activation in response to some extracellular stimuli are reduced. Adjacent to Thr218 and Ile275 is a site where large conformational changes occur in the presence of docking-site peptides derived from p38α substrates and activators. This suggests that TAB1-induced autophosphorylation of p38α results from conformational changes that are similar but unique to those seen in p38α interactions with its substrates and activating kinases.

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