A Conserved Regulatory Module at the C Terminus of the Papillomavirus E1 Helicase Domain Controls E1 Helicase Assembly

ABSTRACTViruses frequently combine multiple activities into one polypeptide to conserve coding capacity. This strategy creates regulatory challenges to ascertain that the combined activities are compatible and do not interfere with each other. The papillomavirus E1 protein, as many other helicases, has the intrinsic ability to form hexamers and double hexamers (DH) that serve as the replicative DNA helicase. However, E1 also has the more unusual ability to generate local melting by forming a double trimer (DT) complex that can untwist the double-stranded origin of DNA replication (ori) DNA in preparation for DH formation. Here we describe a switching mechanism that allows the papillomavirus E1 protein to form these two different kinds of oligomers and to transition between them. We show that a conserved regulatory module attached to the E1 helicase domain blocks hexamer and DH formation and promotes DT formation. In the presence of the appropriate trigger, the inhibitory effect of the regulatory module is relieved and the transition to DH formation can occur.IMPORTANCEThis study provides a mechanistic understanding into how a multifunctional viral polypeptide can provide different, seemingly incompatible activities. A conserved regulatory sequence module attached to the AAA+helicase domain in the papillomavirus E1 protein allows the formation of different oligomers with different biochemical activities.

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Mutations in the WRN Gene in Mice Accelerate Mortality in a p53-Null Background

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.3286-3291.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 3286-3291 ◽

Cited By ~ 125

Author(s):

David B. Lombard ◽

Caroline Beard ◽

Brad Johnson ◽

Robert A. Marciniak ◽

Jessie Dausman ◽

...

Keyword(s):

Mortality Rate ◽

Life Span ◽

Human Disease ◽

Dna Helicase ◽

Premature Aging ◽

Mutant Mice ◽

Helicase Domain ◽

C Terminus ◽

Accelerated Senescence

ABSTRACT Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly,WRN−/− ;p53−/− mice show an increased mortality rate relative toWRN+/− ;p53−/− animals. We consider possible models for the synergy betweenp53 and WRN mutations for the determination of life span.

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The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51

mBio ◽

10.1128/mbio.01192-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 5

Author(s):

Alex Bronstein ◽

Lihi Gershon ◽

Gilad Grinberg ◽

Elisa Alonso-Perez ◽

Martin Kupiec

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Genome Stability ◽

Dna Helicase ◽

Main Role ◽

Helicase Domain ◽

Helicase Activity ◽

C Terminus ◽

Srs2 Helicase

ABSTRACTHomologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. TheSaccharomyces cerevisiaeyeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2’s helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a newSRS2allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of thesrs2(1-850) allele suggest a role for Srs2’s helicase activity in sister chromatid cohesion. Our results also indicate that Srs2’s function becomes more central in diploid cells.IMPORTANCEHomologous recombination (HR) is a key mechanism that repairs damaged DNA. However, this process has to be tightly regulated; failure to regulate it can lead to genome instability. The Srs2 helicase is considered a regulator of HR; it was shown to be able to evict the recombinase Rad51 from DNA. Cells lacking Srs2 exhibit sensitivity to DNA-damaging agents, and in some cases, they display defects in DNA replication. The relative roles of the helicase and Rad51 removal activities of Srs2 in genome stability remain unclear. To address this question, we created a new Srs2 mutant which has only the DNA helicase domain. Our study shows that only the DNA helicase domain is needed to deal with DNA damage and assist in DNA replication during vegetative growth and in meiosis. Thus, our findings shift the view on the role of Srs2 in the maintenance of genome integrity.

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Molecular determinants of KA1 domain-mediated autoinhibition and phospholipid activation of MARK1 kinase

Biochemical Journal ◽

10.1042/bcj20160792 ◽

2017 ◽

Vol 474 (3) ◽

pp. 385-398 ◽

Cited By ~ 14

Author(s):

Ryan P. Emptage ◽

Mark A. Lemmon ◽

Kathryn M. Ferguson

Keyword(s):

Membrane Surface ◽

Kinase Domain ◽

Site Directed Mutagenesis ◽

Anionic Phospholipid ◽

Anionic Phospholipids ◽

Disease States ◽

C Terminus ◽

Regulatory Module ◽

Correct Location ◽

Mechanistic Basis

Protein kinases are frequently regulated by intramolecular autoinhibitory interactions between protein modules that are reversed when these modules bind other ‘activating’ protein or membrane-bound targets. One group of kinases, the MAP/microtubule affinity-regulating kinases (MARKs) contain a poorly understood regulatory module, the KA1 (kinase associated-1) domain, at their C-terminus. KA1 domains from MARK1 and several related kinases from yeast to humans have been shown to bind membranes containing anionic phospholipids, and peptide ligands have also been reported. Deleting or mutating the C-terminal KA1 domain has been reported to activate the kinase in which it is found — also suggesting an intramolecular autoinhibitory role. Here, we show that the KA1 domain of human MARK1 interacts with, and inhibits, the MARK1 kinase domain. Using site-directed mutagenesis, we identify residues in the KA1 domain required for this autoinhibitory activity, and find that residues involved in autoinhibition and in anionic phospholipid binding are the same. We also demonstrate that a ‘mini’ MARK1 becomes activated upon association with vesicles containing anionic phospholipids, but only if the protein is targeted to these vesicles by a second signal. These studies provide a mechanistic basis for understanding how MARK1 and its relatives may require more than one signal at the membrane surface to control their activation at the correct location and time. MARK family kinases have been implicated in a plethora of disease states including Alzheimer's, cancer, and autism, so advancing our understanding of their regulatory mechanisms may ultimately have therapeutic value.

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Both products of the fosB gene, FosB and its short form, FosB/SF, are transcriptional activators in fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5470-5478.1991 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5470-5478

Author(s):

P Dobrazanski ◽

T Noguchi ◽

K Kovary ◽

C A Rizzo ◽

P S Lazo ◽

...

Keyword(s):

Amino Acids ◽

Cell Cycle Progression ◽

Short Form ◽

Constitutive Expression ◽

Inhibitory Effect ◽

Negative Regulator ◽

3T3 Cells ◽

C Terminus ◽

Nih 3T3 ◽

Nih 3T3 Cells

We demonstrate that a member of the fos family, the fosB gene, gives rise to two transcripts by alternative splicing of exon 4, generating two proteins, FosB of 338 amino acids and a short form, FosB/SF, which contains the DNA binding and dimerization domains but not the 101 amino acids of the C terminus. FosB/SF activates an AP-1-chloramphenicol acetyltransferase construct in NIH 3T3 cells, as determined by transient and stable transfections, although more weakly than does FosB. In contrast to FosB, FosB/SF has lost its ability to repress the dyad symmetry element of the c-fos gene. FosB/SF when expressed in excess to FosB can downmodulate the activity of FosB. Constitutive expression of high levels of FosB/SF in NIH 3T3 cells has no significant inhibitory effect in the induction of cell proliferation or cell cycle progression, indicating that FosB/SF is not a negative regulator of cell growth. This conclusion is further confirmed by the observation that the majority of the Jun molecules are complexed with FosB/SF in the FosB/SF-overexpressing cells.

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Inhibition of SARS-CoV-2 in Vero cell cultures by peptide-conjugated morpholino oligomers

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa460 ◽

2020 ◽

Author(s):

Kyle Rosenke ◽

Shanna Leventhal ◽

Hong M Moulton ◽

Susan Hatlevig ◽

David Hawman ◽

...

Keyword(s):

Cell Cultures ◽

Treatment Time ◽

Random Sequence ◽

Antiviral Agents ◽

Inhibitory Effect ◽

Negative Control ◽

Regulatory Sequence ◽

Global Public Health ◽

Genomic Rna ◽

Preclinical Development

Abstract Background As the causative agent of COVID-19, SARS-CoV-2 is a pathogen of immense importance to global public health. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense compounds composed of a phosphorodiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking. Methods Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5′-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method using a 48 h PPMO treatment time. Viral growth was measured with quantitative RT–PCR and TCID50 infectivity assays from experiments where cells received a 5 h PPMO treatment time. Results PPMO designed to base-pair with sequence in the 5′ terminal region or the leader transcription regulatory sequence region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titres by up to 4–6 log10 in cell cultures at 48–72 h post-infection, in a non-toxic and dose-responsive manner. Conclusions The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further preclinical development.

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Prostaglandin E1 inhibits endocytosis in the β-cell endocytosis

Journal of Endocrinology ◽

10.1530/joe-15-0435 ◽

2016 ◽

Vol 229 (3) ◽

pp. 287-294 ◽

Cited By ~ 3

Author(s):

Ying Zhao ◽

Qinghua Fang ◽

Susanne G Straub ◽

Manfred Lindau ◽

Geoffrey W G Sharp

Keyword(s):

Inhibitory Effect ◽

Prostaglandin E ◽

Β Cell ◽

Α Subunit ◽

Capacitance Measurements ◽

Readily Releasable Pool ◽

Whole Cell Patch Clamp ◽

C Terminus ◽

High Level

Prostaglandins inhibit insulin secretion in a manner similar to that of norepinephrine (NE) and somatostatin. As NE inhibits endocytosis as well as exocytosis, we have now examined the modulation of endocytosis by prostaglandin E1 (PGE1). Endocytosis following exocytosis was recorded by whole-cell patch clamp capacitance measurements in INS-832/13 cells. Prolonged depolarizing pulses producing a high level of Ca2+ influx were used to stimulate maximal exocytosis and to deplete the readily releasable pool (RRP) of granules. This high Ca2+ influx eliminates the inhibitory effect of PGE1 on exocytosis and allows specific characterization of the inhibitory effect of PGE1 on the subsequent compensatory endocytosis. After stimulating exocytosis, endocytosis was apparent under control conditions but was inhibited by PGE1 in a Pertussis toxin-sensitive (PTX)-insensitive manner. Dialyzing a synthetic peptide mimicking the C-terminus of the α-subunit of the heterotrimeric G-protein Gz into the cells blocked the inhibition of endocytosis by PGE1, whereas a control-randomized peptide was without effect. These results demonstrate that PGE1 inhibits endocytosis and Gz mediates the inhibition.

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Withaferin A binds covalently to the N-terminal domain of annexin A2

Biological Chemistry ◽

10.1515/hsz-2012-0184 ◽

2012 ◽

Vol 393 (10) ◽

pp. 1151-1163 ◽

Cited By ~ 17

Author(s):

Gabriel Ozorowski ◽

Christopher M. Ryan ◽

Julian P. Whitelegge ◽

Hartmut Luecke

Keyword(s):

Actin Polymerization ◽

Inhibitory Effect ◽

Annexin A2 ◽

Actin Dynamics ◽

Withaferin A ◽

Core Domain ◽

Cancer Pathways ◽

Terminal Domain ◽

C Terminus

Abstract Annexin A2 (AnxA2), a 38-kDa member of the Ca2+-binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.

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A Novel Replicative Enzyme Encoded by the Linear Arthrobacter Plasmid pAL1

Journal of Bacteriology ◽

10.1128/jb.00614-10 ◽

2010 ◽

Vol 192 (19) ◽

pp. 4935-4943 ◽

Cited By ~ 7

Author(s):

Stephan Kolkenbrock ◽

Bianca Naumann ◽

Michael Hippler ◽

Susanne Fetzner

Keyword(s):

Inverted Repeat ◽

Bioinformatic Analysis ◽

Dna Helicase ◽

Terminal Segment ◽

Helicase Domain ◽

Terminal Inverted Repeat ◽

Sequence Motifs ◽

Dna Topoisomerase ◽

Central Regions

ABSTRACT The soil bacterium Arthrobacter nitroguajacolicus Rü61a contains the linear plasmid pAL1, which codes for the degradation of 2-methylquinoline. Like other linear replicons of actinomycetes, pAL1 is characterized by short terminal inverted-repeat sequences and terminal proteins (TPpAL1) covalently attached to its 5′ ends. TPpAL1, encoded by the pAL1.102 gene, interacts in vivo with the protein encoded by pAL1.101. Bioinformatic analysis of the pAL1.101 protein, which comprises 1,707 amino acids, suggested putative zinc finger and topoisomerase-primase domains and part of a superfamily 2 helicase domain in its N-terminal and central regions, respectively. Sequence motifs characteristic of the polymerization domain of family B DNA polymerases are partially conserved in a C-terminal segment. The purified recombinant protein catalyzed the deoxycytidylation of TPpAL1 in the presence of single-stranded DNA templates comprising the 3′-terminal sequence (5′-GCAGG-3′), which in pAL1 forms the terminal inverted repeat, but also at templates with 5′-(G/T)CA(GG/GC/CG)-3′ ends. Enzyme assays suggested that the protein exhibits DNA topoisomerase, DNA helicase, and DNA- and protein-primed DNA polymerase activities. The pAL1.101 protein, therefore, may act as a replicase of pAL1.

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Multifunctional Role of the Pitx2 Homeodomain Protein C-Terminal Tail

Molecular and Cellular Biology ◽

10.1128/mcb.19.10.7001 ◽

1999 ◽

Vol 19 (10) ◽

pp. 7001-7010 ◽

Cited By ~ 79

Author(s):

Brad A. Amendt ◽

Lillian B. Sutherland ◽

Andrew F. Russo

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Inhibitory Effect ◽

Binding Activity ◽

Homeodomain Protein ◽

Rieger Syndrome ◽

Amino Acid Peptide ◽

Dna Binding Activity ◽

C Terminus

ABSTRACT Pitx2 is a newly described bicoid-like homeodomain transcription factor that is defective in Rieger syndrome and shows a striking leftward developmental asymmetry. We have previously shown that Pitx2 (also called Ptx2 and RIEG) transactivates a reporter gene containing abicoid enhancer and synergistically transactivates the prolactin promoter in the presence of the POU homeodomain protein Pit-1. In this report, we focused on the C-terminal region which is mutated in some Rieger patients and contains a highly conserved 14-amino-acid element. Deletion analysis of Pitx2 revealed that the C-terminal 39-amino-acid tail represses DNA binding activity and is required for Pitx2-Pit-1 interaction and Pit-1 synergism. Pit-1 interaction with the Pitx2 C terminus masks the inhibitory effect and promotes increased DNA binding activity. Interestingly, cotransfection of an expression vector encoding the C-terminal 39 amino acids of Pitx2 specifically inhibits Pitx2 transactivation activity. In contrast, the C-terminal 39-amino-acid peptide interacts with Pitx2 to increase its DNA binding activity. These data suggest that the C-terminal tail intrinsically inhibits the Pitx2 protein and that this inhibition can be overcome by interaction with other transcription factors to allow activation during development.

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