scholarly journals Non-Canonical G-quadruplexes cause the hCEB1 minisatellite instability in Saccharomyces cerevisiae

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Aurèle Piazza ◽  
Xiaojie Cui ◽  
Michael Adrian ◽  
Frédéric Samazan ◽  
Brahim Heddi ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structure Function ◽  
Genomic Instability ◽  
Function Analysis ◽  
Structural Features ◽  
Detectable Effect ◽  
Definition Of ◽  
And Function

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids in vitro, but the sequence and structural features dictating their formation and function in vivo remains uncertain. Here we report a structure-function analysis of the complex hCEB1 G4-forming sequence. We isolated four G4 conformations in vitro, all of which bear unusual structural features: Form 1 bears a V-shaped loop and a snapback guanine; Form 2 contains a terminal G-triad; Form 3 bears a zero-nucleotide loop; and Form 4 is a zero-nucleotide loop monomer or an interlocked dimer. In vivo, Form 1 and Form 2 differently account for 2/3rd of the genomic instability of hCEB1 in two G4-stabilizing conditions. Form 3 and an unidentified form contribute to the remaining instability, while Form 4 has no detectable effect. This work underscores the structural polymorphisms originated from a single highly G-rich sequence and demonstrates the existence of non-canonical G4s in cells, thus broadening the definition of G4-forming sequences.

Structure–function analysis of full-length midkine reveals novel residues important for heparin binding and zebrafish embryogenesis

2013 ◽  
Vol 451 (3) ◽  
pp. 407-415 ◽  
Author(s):  
Jackwee Lim ◽  
Sheng Yao ◽  
Martin Graf ◽  
Christoph Winkler ◽  
Daiwen Yang
Keyword(s):  
Function Analysis ◽  
Structural Features ◽  
Full Length ◽  
Domain Growth ◽  
Heparin Binding ◽  
Cellular Mechanisms ◽  
The Individual ◽  
Zebrafish Embryogenesis

Midkine is a heparin-binding di-domain growth factor, implicated in many biological processes as diverse as angiogenesis, neurogenesis and tumorigenesis. Elevated midkine levels reflect poor prognosis for many carcinomas, yet the molecular and cellular mechanisms orchestrating its activity remain unclear. At the present time, the individual structures of isolated half domains of human midkine are known and its functionally active C-terminal half domain remains a popular therapeutic target. In the present study, we determined the structure of full-length zebrafish midkine and show that it interacts with fondaparinux (a synthetic highly sulfated pentasaccharide) and natural heparin through a previously uncharacterized, but highly conserved, hinge region. Mutating six consecutive residues in the conserved hinge to glycine strongly abates heparin binding and midkine embryogenic activity. In contrast with previous in vitro studies, we found that the isolated C-terminal half domain is not active in vivo in embryos. Instead, we have demonstrated that the N-terminal half domain is needed to enhance heparin binding and mediate midkine embryogenic activity surprisingly in both heparin-dependent and -independent manners. Our findings provide new insights into the structural features of full-length midkine relevant for embryogenesis, and unravel additional therapeutic routes targeting the N-terminal half domain and conserved hinge.

scholarly journals Structure/Function Analysis of the Vaccinia Virus F18 Phosphoprotein, an Abundant Core Component Required for Virion Maturation and Infectivity

2010 ◽  
Vol 84 (13) ◽  
pp. 6846-6860 ◽  
Author(s):  
Nadi T. Wickramasekera ◽  
Paula Traktman
Keyword(s):  
Amino Acids ◽  
Structure Function ◽  
Protein Interactions ◽  
Function Analysis ◽  
Aromatic Amino Acids ◽  
Protein Protein Interactions ◽  
Core Component ◽  
Virion Morphogenesis

ABSTRACT Poxvirus virions, whose outer membrane surrounds two lateral bodies and a core, contain at least 70 different proteins. The F18 phosphoprotein is one of the most abundant core components and is essential for the assembly of mature virions. We report here the results of a structure/function analysis in which the role of conserved cysteine residues, clusters of charged amino acids and clusters of hydrophobic/aromatic amino acids have been assessed. Taking advantage of a recombinant virus in which F18 expression is IPTG (isopropyl-β-d-thiogalactopyranoside) dependent, we developed a transient complementation assay to evaluate the ability of mutant alleles of F18 to support virion morphogenesis and/or to restore the production of infectious virus. We have also examined protein-protein interactions, comparing the ability of mutant and WT F18 proteins to interact with WT F18 and to interact with the viral A30 protein, another essential core component. We show that F18 associates with an A30-containing multiprotein complex in vivo in a manner that depends upon clusters of hydrophobic/aromatic residues in the N′ terminus of the F18 protein but that it is not required for the assembly of this complex. Finally, we confirmed that two PSSP motifs within F18 are the sites of phosphorylation by cellular proline-directed kinases in vitro and in vivo. Mutation of both of these phosphorylation sites has no apparent impact on virion morphogenesis but leads to the assembly of virions with significantly reduced infectivity.

scholarly journals Structure-function relationships in mitochondrial transcriptional condensates

2022 ◽  
Author(s):  
Marina Feric ◽  
Azadeh Sarfallah ◽  
Furqan Dar ◽  
Dmitry Temiakov ◽  
Rohit V Pappu ◽  
...  
Keyword(s):  
Structure Function ◽  
Computational Modelling ◽  
In Vivo Studies ◽  
Bulk Solution ◽  
The Core ◽  
Functional Consequences ◽  
Cellular Machinery ◽  
And Function

Phase separation organizes many membraneless structures in cells. The functional consequences of concentrating cellular machinery into biomolecular condensates, however, is largely unclear. Here, we use in vitro reconstitutions, in vivo studies, and computational modelling to uncover structure-function relationships of mitochondrial (mt-) transcriptional condensates. In vitro, we find that the core mt-transcription machinery — consisting of POLRMT, TFAM, TFB2M, and DNA — forms viscoelastic, multi-phasic condensates. Strikingly, the rates of condensate-mediated transcription are considerably lower than equivalent reactions in bulk solution. Dampened rates are associated with reduced diffusivities of components that become kinetically arrested in non-equilibrium, vesicular condensates. Perturbation of mt-components in vivo and computational simulations recapitulate the transcription-dependent reorganizations observed in vitro. Our findings demonstrate close, bidirectional interdependence between structure and function of transcriptional condensates.

scholarly journals Rad53 Checkpoint Kinase Phosphorylation Site Preference Identified in the Swi6 Protein of Saccharomyces cerevisiae

2003 ◽  
Vol 23 (10) ◽  
pp. 3405-3416 ◽  
Author(s):  
Julia M. Sidorova ◽  
Linda L. Breeden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Phosphorylation Site ◽  
Site Preference ◽  
Phosphorylation Sites ◽  
Checkpoint Kinase ◽  
Checkpoint Signaling ◽  
And Function

ABSTRACT Rad53 of Saccharomyces cerevisiae is a checkpoint kinase whose structure and function are conserved among eukaryotes. When a cell detects damaged DNA, Rad53 activity is dramatically increased, which ultimately leads to changes in DNA replication, repair, and cell division. Despite its central role in checkpoint signaling, little is known about Rad53 substrates or substrate specificity. A number of proteins are implicated as Rad53 substrates; however, the evidence remains indirect. Previously, we have provided evidence that Swi6, a subunit of the Swi4/Swi6 late-G1-specific transcriptional activator, is a substrate of Rad53 in the G1/S DNA damage checkpoint. In the present study we identify Rad53 phosphorylation sites in Swi6 in vitro and demonstrate that at least one of them is targeted by Rad53 in vivo. Mutations in these phosphorylation sites in Swi6 shorten but do not eliminate the Rad53-dependent delay of the G1-to-S transition after DNA damage. We derive a consensus for Rad53 site preference at positions −2 and +2 (−2/+2) and identify its potential substrates in the yeast proteome. Finally, we present evidence that one of these candidates, the cohesin complex subunit Scc1 undergoes DNA damage-dependent phosphorylation, which is in part dependent on Rad53.

scholarly journals Structure-guided disruption of pseudopilus tip inhibits Type II secretion in Pseudomonas aeruginosa

2018 ◽  
Author(s):  
Zongchao Jia ◽  
Yichen Zhang ◽  
Frederick Faucher ◽  
Wenwen Zhang ◽  
Shu Wang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Function Analysis ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Core Complex ◽  
Wide Range ◽  
Interaction Interface ◽  
And Function

Pseudomonas aeruginosa utilizes the Type II secretion system (T2SS) to translocate a wide range of large, structured protein virulence factors through the periplasm to the extracellular environment for infection. In the T2SS, five pseudopilins assemble into the pseudopilus that acts as a piston to extrude exoproteins out of cells. Through structure determination of the pseudopilin complexes of XcpVWX and XcpVW and function analysis, we have confirmed that two minor pseudopilins, XcpV and XcpW, constitute a core complex indispensable to the pseudopilus tip. The absence of either XcpV or -W resulted in the non-functional T2SS. Our small-angle X-ray scattering experiment for the first time revealed the architecture of the entire pseudopilus tip and established the working model. Based on the interaction interface of complexes, we have developed inhibitory peptides. The structure-based peptides not only disrupted of the XcpVW core complex and the entire pseudopilus tip in vitro but also inhibited the T2SS in vivo. More importantly, these peptides effectively reduced the virulence of P. aeruginosa towards Caenorhabditis elegans.

Structure-Function Analysis of RNAs Generated by In Vivo and In Vitro Selection

2002 ◽  
Vol 216 (2) ◽  
Author(s):  
Andreas Schwienhorst
Keyword(s):  
Structure Function ◽  
Significant Role ◽  
In Vitro Selection ◽  
Function Analysis ◽  
Darwinian Evolution

Today, the concept of Darwinian evolution plays a significant role in studying structure-function relationships concerning known molecules and in helping to design previously unknown molecules with desired functionalities. Results from

scholarly journals Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications

2021 ◽  
Vol 17 ◽  
pp. 1641-1688
Author(s):  
Nikita Brodyagin ◽  
Martins Katkevics ◽  
Venubabu Kotikam ◽  
Christopher A Ryan ◽  
Eriks Rozners
Keyword(s):  
Biomedical Applications ◽  
Structural Features ◽  
Full Potential ◽  
Binding Modes ◽  
Current State ◽  
High Doses ◽  
And Function ◽  
Dramatic Departure

Peptide nucleic acid (PNA) is arguably one of the most successful DNA mimics, despite a most dramatic departure from the native structure of DNA. The present review summarizes 30 years of research on PNA’s chemistry, optimization of structure and function, applications as probes and diagnostics, and attempts to develop new PNA therapeutics. The discussion starts with a brief review of PNA’s binding modes and structural features, followed by the most impactful chemical modifications, PNA enabled assays and diagnostics, and discussion of the current state of development of PNA therapeutics. While many modifications have improved on PNA’s binding affinity and specificity, solubility and other biophysical properties, the original PNA is still most frequently used in diagnostic and other in vitro applications. Development of therapeutics and other in vivo applications of PNA has notably lagged behind and is still limited by insufficient bioavailability and difficulties with tissue specific delivery. Relatively high doses are required to overcome poor cellular uptake and endosomal entrapment, which increases the risk of toxicity. These limitations remain unsolved problems waiting for innovative chemistry and biology to unlock the full potential of PNA in biomedical applications.

Sign in / Sign up

Export Citation Format

Share Document