GPR34 activation potentially bridges lymphoepithelial lesion to genesis of salivary gland MALT lymphoma

Blood ◽  
2021 ◽  
Author(s):  
Boguslawa Korona ◽  
Dagmara Korona ◽  
Wanfeng Zhao ◽  
Andrew Wotherspoon ◽  
Ming-Qing Du
Keyword(s):  
Salivary Gland ◽  
Malt Lymphoma ◽  
Single Copy ◽  
In Vitro Assays ◽  
Wild Type ◽  
Phospholipase A1 ◽  
Genetic Changes ◽  
C Terminus ◽  
Truncation Mutant

GPR34 translocation and mutation are specifically associated with salivary gland MALT lymphoma (SG-MALT-Lymphoma). Majority of GPR34 mutations are clustered in its C-terminus, resulting in truncated proteins lacking the phosphorylation motif important for receptor desensitization. It is unclear why GPR34 genetic changes associate with SG-MALT-Lymphoma and how these mutations contribute to the lymphoma development. We generated isogenic Flp-InTRex293 cell lines that stably expressed a single copy of GPR34 or its various mutants, and performed a range of in vitro assays. We showed that the GPR34 Q340X truncation, but not R84H and D151A mutants conferred a significantly increased resistance to apoptosis, and greater transforming potential than the GPR34 wild type. The GPR34 truncation mutant had a significantly delayed internalization than the wild type following ligand (lysophosphatidylserine) stimulation. Among 9 signaling pathways examined, the GPR34 Q340X truncation, to a lesser extent the D151A mutant, significantly activated CRE, NFkB and AP1 reporter activities, particularly in the presence of ligand stimulation. We further demonstrated enhanced activities of phospholipase-A1/2 in the culture supernatant of Flp-InTRex293 cells that expressed the GPR34 Q340X mutant, and their potential to catalyze the synthesis of lysophosphatidylserine from phosphatidylserine. Importantly, phospholipase-A1 was abundantly expressed in the duct epithelium of salivary glands and those involved in lymphoepithelial lesions (LELs). Our findings advocate a model of paracrine stimulation of malignant B-cells via GPR34, in which PLA is released by LELs, and hydrolyzes the phosphatidylserine exposed on apoptotic cells, generating lysophosphatidylserine, the ligand for GPR34. Thus, GPR34 activation potentially bridges LELs to genesis of SG-MALT-Lymphoma.

scholarly journals Subforms and In Vitro Reconstitution of the NAD-Reducing Hydrogenase of Alcaligenes eutrophus

1998 ◽  
Vol 180 (5) ◽  
pp. 1023-1029 ◽  
Author(s):  
Christian Massanz ◽  
Silke Schmidt ◽  
Bärbel Friedrich
Keyword(s):  
Alcaligenes Eutrophus ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Monomeric Form ◽  
Structural Genes ◽  
Wild Type ◽  
Catalytic Function ◽  
C Terminus ◽  
In Vitro Reconstitution ◽  
Reducing Activity

The cytoplasmic, NAD-reducing hydrogenase (SH) of Alcaligenes eutrophus H16 is a heterotetrameric enzyme which contains several cofactors and undergoes a complex maturation during biogenesis. HoxH is the Ni-carrying subunit, and together with HoxY it forms the hydrogenase dimer. HoxF and HoxU represent the flavin-containing diaphorase moiety, which is closely related to NADH:ubiquinone oxidoreductase and mediates NADH oxidation. A variety of mutations were introduced into the four SH structural genes to obtain mutant enzymes composed of monomeric and dimeric forms. A deletion removing most ofhoxF, hoxU, and hoxY led to the expression of a HoxH monomer derivative which was proteolytically processed at the C terminus like the wild-type polypeptide. While the hydrogenase dimer, produced by a strain deleted of hoxF andhoxU, displayed H2-dependent dye-reducing activity, the monomeric form did not mediate the activation of H2, although nickel was incorporated into HoxH. Deletion ofhoxH and hoxY led to the production of HoxFU dimers which displayed NADH:oxidoreductase activity. Mixing the hydrogenase and the diaphorase moieties in vitro reconstituted the structure and catalytic function of the SH holoenzyme.

scholarly journals Genetic Analysis of theEscherichia coliFtsZ·ZipA Interaction in the Yeast Two-hybrid System

2001 ◽  
Vol 276 (15) ◽  
pp. 11980-11987 ◽  
Author(s):  
Steven A. Haney ◽  
Elizabeth Glasfeld ◽  
Cynthia Hale ◽  
David Keeney ◽  
Zhizhen He ◽  
...  
Keyword(s):  
Hybrid System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Conserved Sequence ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Two Hybrid

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division inEscherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZD373G) identified eight different changes at two residues within this sequence.In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZD373Gfailed to interact. Two mutant proteins examined restored this interaction significantly.In vivo, the alleles tested are significantly more toxic than the wild typeftsZand cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZD373G, no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals Substitutions in the Periplasmic Domain of Low-Abundance Chemoreceptor Trg That Induce or Reduce Transmembrane Signaling: Kinase Activation and Context Effects

2001 ◽  
Vol 183 (2) ◽  
pp. 671-679 ◽  
Author(s):  
Bryan D. Beel ◽  
Gerald L. Hazelbauer
Keyword(s):  
Context Effects ◽  
High Abundance ◽  
In Vitro Assays ◽  
Wild Type ◽  
Kinase Activation ◽  
Interaction Sites ◽  
Natural Enzyme ◽  
Signaling Receptors

ABSTRACT We extended characterization of mutational substitutions in the ligand-binding region of Trg, a low-abundance chemoreceptor ofEscherichia coli. Previous investigations using patterns of adaptational methylation in vivo led to the suggestion that one class of substitutions made the receptor insensitive, reducing ligand-induced signaling, and another mimicked ligand occupancy, inducing signaling in the absence of ligand. We tested these deductions with in vitro assays of kinase activation and found that insensitive receptors activated the kinase as effectively as wild-type receptors and that induced-signaling receptors exhibited the low level of kinase activation characteristic of occupied receptors. Differential activation by the two mutant classes was not dependent on high-abundance receptors. Cellular context can affect the function of low-abundance receptors. Assays of chemotactic response and adaptational modification in vivo showed that increasing cellular dosage of mutant forms of Trg to a high-abundance level did not significantly alter phenotypes, nor did the presence of high-abundance receptors significantly correct phenotypic defects of reduced-signaling receptors. In contrast, defects of induced-signaling receptors were suppressed by the presence of high-abundance receptors. Grafting the interaction site for the adaptational-modification enzymes to the carboxyl terminus of induced-signaling receptors resulted in a similar suppression of phenotypic defects of induced-signaling receptors, implying that high-abundance receptors could suppress defects in induced-signaling receptors by providing their natural enzyme interaction sites intrans in clusters of suppressing and suppressed receptors. As in the case of cluster-related functional assistance provided by high-abundance receptors for wild-type low-abundance receptors, suppression by high-abundance receptors of phenotypic defects in induced-signaling forms of Trg involved assistance in adaptation, not signaling.

An in vitro model for genetic changes associated with the adenomacarcinoma sequence in salivary gland tumors

1992 ◽  
Vol 63 (2) ◽  
pp. 118
Author(s):  
Bernd Kazmierczak ◽  
Brita Thode ◽  
Sabine Bartnitzke ◽  
Jörn Bullerdiek ◽  
Werner Schloot
Keyword(s):  
Salivary Gland ◽  
In Vitro Model ◽  
Salivary Gland Tumors ◽  
Genetic Changes ◽  
Vitro Model

scholarly journals Leptospiral LruA Is Required for Virulence and Modulates an Interaction with Mammalian Apolipoprotein AI

2013 ◽  
Vol 81 (10) ◽  
pp. 3872-3879 ◽  
Author(s):  
Kunkun Zhang ◽  
Gerald L. Murray ◽  
Torsten Seemann ◽  
Amporn Srikram ◽  
Thanatchaporn Bartpho ◽  
...  
Keyword(s):  
Serum Protein ◽  
Serum Proteins ◽  
Acute Infection ◽  
Cellular Interactions ◽  
Wild Type ◽  
Hamster Model ◽  
Content Type ◽  
Truncation Mutant ◽  
Separation Of Proteins

ABSTRACTLeptospirosis is a worldwide zoonosis caused by spirochetes of the genusLeptospira. While understanding of pathogenesis remains limited, the development of mutagenesis inLeptospirahas provided a powerful tool for identifying novel virulence factors. LruA is a lipoprotein that has been implicated in leptospiral uveitis as a target of the immune response. In this study, twolruAmutants, M754 and M765, generated by transposon mutagenesis fromLeptospira interrogansserovar Manilae, were characterized. In M754, the transposon inserted in the middle oflruA, resulting in no detectable expression of LruA. In M765, the transposon inserted toward the 3′ end of the gene, resulting in expression of a truncated protein. LruA was demonstrated to be on the cell surface in M765 and the wild type (WT). M754, but not M765, was attenuated in a hamster model of acute infection. A search for differential binding to human serum proteins identified a serum protein of around 30 kDa bound to the wild type and the LruA deletion mutant (M754), but not to the LruA truncation mutant (M765). Two-dimensional separation of proteins from leptospiral cells incubated with guinea pig serum identified the 28-kDa apolipoprotein A-I (ApoA-I) as a major mammalian serum protein that bindsLeptospirain vitro. Interestingly, M754 (with no detectable LruA) bound more ApoA-I than did the LruA-expressing strains Manilae wild type and M765. Our data thus identify LruA as a surface-exposed leptospiral virulence factor that contributes to leptospiral pathogenesis, possibly by modulating cellular interactions with serum protein ApoA-I.

scholarly journals Cnu, a Novel oriC-Binding Protein of Escherichia coli

2005 ◽  
Vol 187 (20) ◽  
pp. 6998-7008 ◽  
Author(s):  
Myung Suk Kim ◽  
Sung-Hun Bae ◽  
Sang Hoon Yun ◽  
Hee Jung Lee ◽  
Sang Chun Ji ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid Identity ◽  
Replication Initiation ◽  
In Vitro Assays ◽  
Wild Type ◽  
Genetic Method ◽  
Dna Replication Initiation ◽  
Pair Sequence

ABSTRACT We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

scholarly journals Conserved C-Terminal Threonine of Hepatitis C Virus NS3 Regulates Autoproteolysis and Prevents Product Inhibition

2004 ◽  
Vol 78 (2) ◽  
pp. 700-709 ◽  
Author(s):  
Wenyan Wang ◽  
Frederick C. Lahser ◽  
MinKyung Yi ◽  
Jacquelyn Wright-Minogue ◽  
Ellen Xia ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
In Vitro Transcription ◽  
Product Inhibition ◽  
Cleavage Sites ◽  
Wild Type ◽  
Genome Sequences ◽  
Mutant Proteins ◽  
C Terminus

ABSTRACT Inspection of over 250 hepatitis C virus (HCV) genome sequences shows that a threonine is strictly conserved at the P1 position in the NS3-NS4A (NS3-4A) autoproteolysis junction, while a cysteine is maintained as the P1 residue in all of the putative trans cleavage sites (NS4A-4B, NS4B-5A, and NS5A-5B). To understand why T631 is conserved at the NS3-4A junction of HCV, a series of in vitro transcription-translation studies were carried out using wild-type and mutant (T631C) NS3-4A constructs bearing native, truncated, and mutant NS4A segments. The autocleavage of the wild-type junction was found to be dependent on the presence of the central cofactor domain of NS4A (residues 21 to 34). In contrast, all NS3-4A T631C mutant proteins underwent self-cleavage even in the absence of the cofactor. Subgenomic replicons derived from the Con1 strain of HCV and bearing the T631C mutation showed reduced levels of colony formation in transfection studies. Similarly, replicons derived from a second genotype 1b virus, HCV-N, demonstrated a comparable reduction in replication efficiency in transient-transfection assays. These data suggest that the threonine is conserved at position 631 because it serves two functions: (i) to slow processing at the NS3-4A cleavage site, ensuring proper intercalation of the NS4A cofactor with NS3 prior to polyprotein scission, and (ii) to prevent subsequent product inhibition by the NS3 C terminus.

scholarly journals Disruption of the ech42 (Endochitinase-Encoding) Gene Affects Biocontrol Activity in Trichoderma harzianum P1

1999 ◽  
Vol 12 (5) ◽  
pp. 419-429 ◽  
Author(s):  
S. L. Woo ◽  
B. Donzelli ◽  
F. Scala ◽  
R. Mach ◽  
G. E. Harman ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Trichoderma Harzianum ◽  
Pythium Ultimum ◽  
Single Copy ◽  
Wild Type ◽  
Gene Encoding ◽  
Culture Filtrates ◽  
In Vitro Antifungal Activity ◽  
Better Than

The biocontrol strain P1 of Trichoderma harzianum was genetically modified by targeted disruption of the single-copy ech42 gene encoding for the secreted 42-kDa endochitinase (CHIT42). Stable mutants in which ech42 was interrupted, and unable to produce CHIT42, were obtained and characterized. These mutants lacked the ech42 transcript, the protein, and endochitinase activity in culture filtrates, and they were unable to clear a medium containing colloidal chitin. Other chitinolytic and glucanolytic enzymes expressed during mycoparasitism were not affected by the disruption of ech42. The disrupted mutant D11 grew and sporulated similarly to the wild type. In vitro antifungal activity of the ech42 disruptant culture filtrates against Botrytis cinerea and Rhizoctonia solani was reduced about 40%, compared with wild type; antifungal activity was fully restored by adding an equivalent amount of CHIT42 as secreted by P1. The mutant exhibited the same biocontrol effect against Pythium ultimum as strain P1, but the antagonism against B. cinerea on bean leaves by the mutant was significantly reduced (33% less biocontrol), compared with strain P1. Conversely, the endochitinase-deficient mutant performed better than the wild type (16% improvement of survival) in biocontrol experiments in soil infested with the soilborne fungus R. solani. These results indicate that the antagonistic interaction between the T. harzianum strain and various fungal hosts is based on different mechanisms.

scholarly journals RepB C-terminus mutation of an ori pRi vector affects plasmid copy number inAgrobacteriumand transgene copy number in plants

2018 ◽  
Author(s):  
Zarir Vaghchhipawala ◽  
Sharon Radke ◽  
Ervin Nagy ◽  
Mary L. Russell ◽  
Susan Johnson ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Copy Number ◽  
Binary Vector ◽  
Single Copy ◽  
Plasmid Copy Number ◽  
Plant Production ◽  
Silent Mutation ◽  
Wild Type ◽  
Plasmid Copy ◽  
C Terminus

AbstractA nativerepABCreplication origin, ori pRi, was previously reported as a single copy plasmid inAgrobacterium tumefaciensand can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors forAgrobacterium-mediatedtransformation. A high copy ori pRi variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::RirepABCoperon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type oriRi binary vector showed thatAgrobacteriumcells with the RepBY299Hmutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299Hmutation on transformation and quality plant production, the RepBY299Hmutated ori pRi binary vector was compared with the original wild-type ori pRi binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy ori pRi with the RepBY299Hmutation inAgrobacteriumcells lost the advantage of generating high frequency single copy, backbone-free transgenic plants compared to using the single copy wild-type ori pRi binary vector.

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