Abstract 1042: Viral Chemokine Modulating Protein, M-T7, Interrupts Chemokine : Glycosaminoglycan (GAG) Interaction: M-T7 Inhibitory Activity is Dependent upon Y46 and V210 aminoacid residues

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Erbin Dai ◽  
Dana McIvor ◽  
Liying Liu ◽  
Ganesh Munaswamy-Ramanujam ◽  
Yunming Sun ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Invasion ◽  
Inhibitory Activity ◽  
Active Sites ◽  
Point Mutations ◽  
List Type ◽  
Wild Type ◽  
Monocyte Activation ◽  
Cc Chemokine ◽  
Plaque Growth

Background: Chemokines bind to glycosaminoglycans (GAGs) forming gradients that direct inflammatory cell invasion. The viral chemokine modulating protein (CMP), MT-7 binds the C terminal, GAG-binding domain of chemokines and has been previously reported to significantly reduce cell invasion and plaque growth in rat aortic and renal transplant models. Two other viral CMPs, M-T1 and M3 CMPs bind the N terminal domain of chemokines that bind to cell surface receptors. To determine the role of CC chemokine receptor 2 (CCR2) and GAGs for M-T7 anti-inflammatory activity, effects of M-T7 on plaque growth were assessed after mouse CCR2 deficient (CCR2−/−) or GAG deficient (NDST1−/−) aortic allograft transplant. Mononuclear cell migration in response to MCP-1 or RANTES into mouse ascites was also tested. Active sites necessary for M-T7 inhibition of chemokine function and monocyte activation, were assessed by infusion of in the mouse cell migration and human monocyte membrane fluidity assays. Results: M-T7 significantly reduced cell migration and intimal hyperplasia in wild type CCR2+/+ (p<0.009), and CCR2−/− aortic transplants (p<0.026). M-T1 and M3 inhibited cell invasion and plaque in CCR2+/+, but not CCR2−/− mice. M-T7 inhibited plaque growth and CC chemokine (MCP-1 and RANTES)-induced cell migration in wild type mice (P<0.01), but not in NDST1−/− mice (P=0.34). Selected M-T7 point mutations Ty (Y)46A, and Val (V) 210A no longer block chemokine-induced cell migration nor monocyte activation, whereas Asn (N) 40, Asn (N) 63 and Val (V)129 retain inhibitory activity. Conclusions: M-T7 but not M-T1 nor M3, blocks cell migration and plaque growth in CCR2 deficient (CCR2−/−) mouse aortic transplant models. M-T7 loses the ability to block cell migration and plaque growth in NDST1−/−, GAG (heparan sulfate) deficient mice. Point mutations Tyr46 and Val 210 lack inflammatory for mouse and human inflammatory monocyte responses indicating that these amino acid residues on the M-T7 CMP protein are required for inhibitory activity.

scholarly journals Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

2001 ◽  
Vol 82 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Andres Merits ◽  
Lidia Vasiljeva ◽  
Tero Ahola ◽  
Leevi Kääriäinen ◽  
Petri Auvinen
Keyword(s):  
Mammalian Cells ◽  
Active Sites ◽  
Insect Cells ◽  
Semliki Forest Virus ◽  
Point Mutations ◽  
Proteolytic Processing ◽  
Wild Type ◽  
In Trans ◽  
Polyprotein Precursor

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

Study Of Activity Of E6201, a Dual FLT3 and MEK Inhibitor, In Acute Myelogenous Leukemia With FLT3 Or RAS Mutation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2683-2683 ◽  
Author(s):  
Weiguo Zhang ◽  
Gautam Borthakur ◽  
Chen Gao ◽  
Ye Chen ◽  
Yong S. Lan ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Inhibitory Activity ◽  
Point Mutations ◽  
Myelogenous Leukemia ◽  
Vehicle Group ◽  
Wild Type ◽  
Ras Mutation ◽  
Induced Apoptosis ◽  
Resistant Cells

Abstract Internal tandem duplication (ITD) or point mutation of Fms-like tyrosine kinase 3 (FLT3) and N/KRAS mutations in patients with acute myeloid leukemia (AML) lead to aberrant activation of FLT3 and/or RAS–mitogen-activated protein kinase (MAPK) pathways and are associated with poor prognosis (Kottaridis et al, Leuk Lymphoma Vol. 44:905, 2003; Thiede et al, Blood Vol. 99;4326, 2002). Therapy with inhibitors targeting these pathways individually may at best result in short lasting responses in the appropriate mutational context (Borthakur et al, Haematologica Vol. 96:62, 2011; Cortes et al, Blood Vol. 114:636a, 2009). Persistent activation of MEK/ERK signaling pathway is seen in cells resistant to FLT3-ITD inhibitor sorafenib that harbor acquired point mutations of FLT3 in tyrosine kinase domains in addition to ITD mutation (Moore et al, Leukemia Vol. 26:1462, 2012). E6201 is a MEK1/FLT3 dual inhibitor with inhibitory activity in low nanomolar concentrations against both targets. We tested E6201 against AML cells including FLT3-inhibitor resistant cells, AML patient samples and investigated its efficacy in murine AML model. E6201 inhibited cell growth and induced apoptosis in AML cells with FLT3 ITD mutations (including sorafenib-resistant cells harboring ITD plus N676D/Y842C point mutations) at nanomolar levels, and showed 600 to 1000-fold more selective activity against cells with FLT3-ITD mutations than those with FLT3-WT (IC50s 0.003µM, 0.005 and 0.002µM, respectively, in Ba/F3-ITD and FLT3-ITD mutant MOLM13 and MV4-11cells compared to 3.18µM in Ba/F3-FLT3-WT cells). In addition, OCI/AML3 cells [FLT3 and RAS wild-type(WT)], which have high basal p-ERK level and are resistant to most of chemotherapeutic drugs, were sensitive to E6201 (IC50 = 0.037µM). Consistent with its MEK1 inhibitory activity, E6201 was more active against NRAS mutation carrying OCI/AML3 and MV4-11 cells than their NRAS-WT isogenic cells. E6201-induced apoptosis appears to be p53 dependent as p53-wild-type OCI/AML3 and MOLM13 cells were significantly more sensitive compared to their paired p53-knockdown cells. EC50 of E6201 was at sub-micromolar levels in all 5 FLT3-ITD mutant primary AML samples, which included one with FLT3-ITD/RAS dual mutation. NOG mice bearing xenografts of MOLM13-Luc-GFP (FLT3-ITD mutated) cells were treated with E6201 i.v. starting on day 5 after leukemia cell injection until day 21 on a twice-per-week schedule. Bioluminescence imaging revealed that the tumor burden (mean luminescence) was reduced (3.1 x 106 and 2.7 x 106vs. 5.6 x 106 Photons/sec, p< .01 in 20mg/kg and 40mg/kg groups compared with vehicle group)(Fig 1) and histologically leukemia cells infiltrations were profoundly reduced in the bone marrows, spleens, livers and lungs on Day 9 after first drug treatment. Additionally, the median survival was modestly extended from 16 days of vehicle-treated mice to 18 days of E6201-treated mice (P <0.01). Mechanistically, E6201 significantly suppressed p-FLT3 and p-ERK in all tested FLT3 mutant AML cell lines and p53 wild-type OCI/AML3 cells. In addition, decrease of Bcl-xL and Mcl-1 levels and increase of cleaved-caspase-3 was observed in all FLT3 mutant cell lines after treatment with E6201 for 24 hours. In conclusion, E6201 is active against AML cells with FLT3 and/or RAS mutation. A clinical trial is in development in FLT3 and/or RAS mutant AML. Disclosures: Borthakur: Eisai, Inc.: Research Funding. Nomoto:Eisai, Inc.: Employment. Zhao:Eisai, Inc.: Employment.

scholarly journals Substrate Affinity and Specificity of the ScSth1p Bromodomain Are Fine-Tuned for Versatile Histone Recognition

2019 ◽  
Author(s):  
Bartlomiej J. Blus ◽  
Hideharu Hashimoto ◽  
Hyuk-Soo Seo ◽  
Aleksandra Krolak ◽  
Erik W. Debler
Keyword(s):  
Binding Sites ◽  
Biological Function ◽  
Point Mutations ◽  
Substrate Affinity ◽  
List Type ◽  
Wild Type ◽  
Wide Range ◽  
Lysine Residues ◽  
Loop Regions ◽  
Peptide Binding Affinity

SummaryBromodomains recognize a wide range of acetylated lysine residues in histones and other nuclear proteins. Substrate specificity is critical for their biological function and arises from unique acetyl-lysine binding sites formed by variable loop regions. Here, we analyzed substrate affinity and specificity of the yeast ScSth1p bromodomain, an essential component of the “Remodels the Structure of Chromatin” complex, and found that the wild-type bromodomain preferentially recognizes H3K14ac and H4K20ac peptides. Mutagenesis studies—guided by our crystal structure determined at 2.7 Å resolution—revealed loop residues Ser1276 and Trp1338 as key determinants for such interactions. Strikingly, point mutations of each of these residues substantially increased peptide binding affinity and selectivity, respectively. Our data demonstrate that the ScSth1p bromodomain is not optimized for binding to an individual acetylation mark, but fine-tuned for interactions with several such modifications, consistent with the versatile and multivalent nature of histone recognition by reader modules such as bromodomains.HighlightsThe ScSth1p bromodomain preferentially recognizes H3K14ac and H4K20ac peptidesSer1276 and Trp1338 are key determinants of substrate affinity and specificityMutations of these residues drastically increase substrate affinity and specificityThe ScSth1p bromodomain is fine-tuned for promiscuous histone tail recognitionGraphical Abstract

Analysis of Inhibitory Activity and Antineoplastic Effect of Wild Type rBTI and Its Mutants*

2010 ◽  
Vol 37 (6) ◽  
pp. 654-661 ◽  
Author(s):  
Xin TIAN ◽  
Chen LI ◽  
Yu-Ying LI ◽  
Zhuan-Hua WANG
Keyword(s):  
Inhibitory Activity ◽  
Wild Type ◽  
Antineoplastic Effect

scholarly journals EB1 Restricts Breast Cancer Cell Invadopodia Formation and Matrix Proteolysis via FAK

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 388
Author(s):  
Brice Chanez ◽  
Kevin Ostacolo ◽  
Ali Badache ◽  
Sylvie Thuault
Keyword(s):  
Breast Cancer ◽  
Cancer Cell ◽  
Cell Invasion ◽  
Focal Adhesions ◽  
Matrix Degradation ◽  
Cancer Cell Invasion ◽  
Wild Type ◽  
Spatial Regulation ◽  
Invadopodia Formation

Regulation of microtubule dynamics by plus-end tracking proteins (+TIPs) plays an essential role in cancer cell migration. However, the role of +TIPs in cancer cell invasion has been poorly addressed. Invadopodia, actin-rich protrusions specialized in extracellular matrix degradation, are essential for cancer cell invasion and metastasis, the leading cause of death in breast cancer. We, therefore, investigated the role of the End Binding protein, EB1, a major hub of the +TIP network, in invadopodia functions. EB1 silencing increased matrix degradation by breast cancer cells. This was recapitulated by depletion of two additional +TIPs and EB1 partners, APC and ACF7, but not by the knockdown of other +TIPs, such as CLASP1/2 or CLIP170. The knockdown of Focal Adhesion Kinase (FAK) was previously proposed to similarly promote invadopodia formation as a consequence of a switch of the Src kinase from focal adhesions to invadopodia. Interestingly, EB1-, APC-, or ACF7-depleted cells had decreased expression/activation of FAK. Remarkably, overexpression of wild type FAK, but not of FAK mutated to prevent Src recruitment, prevented the increased degradative activity induced by EB1 depletion. Overall, we propose that EB1 restricts invadopodia formation through the control of FAK and, consequently, the spatial regulation of Src activity.

scholarly journals CC Chemokine Ligand 7 Derived from Cancer-Stimulated Macrophages Promotes Ovarian Cancer Cell Invasion

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2745
Author(s):  
Miran Jeong ◽  
Yi-Yue Wang ◽  
Ju-Yeon Choi ◽  
Myong-Cheol Lim ◽  
Jung-Hye Choi
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Cell Invasion ◽  
Ovarian Cancer Cell ◽  
Ovarian Cancer Cells ◽  
Human Ovarian Cancer ◽  
Cancer Cell Invasion ◽  
Cc Chemokine ◽  
Chemokine Ligand

In the tumor microenvironment, macrophages have been suggested to be stimulated by tumor cells, becoming tumor-associated macrophages that promote cancer development and progression. We examined the effect of these macrophages on human ovarian cancer cell invasion and found that conditioned medium of macrophages stimulated by ovarian cancer cells (OC-MQs) significantly increased cell invasion. CC chemokine ligand 7 (CCL7) expression and production were significantly higher in OC-MQs than in the control macrophages. Peritoneal macrophages from patients with ovarian cancer showed higher CCL7 expression levels than those from healthy controls. Inhibition of CCL7 using siRNA and neutralizing antibodies reduced the OC-MQ-CM-induced ovarian cancer cell invasion. CC chemokine receptor 3 (CCR3) was highly expressed in human ovarian cancer cells, and a specific inhibitor of this receptor reduced the OC-MQ-CM-induced invasion. Specific signaling and transcription factors were associated with enhanced CCL7 expression in OC-MQs. CCL7-induced invasion required the expression of matrix metalloproteinase 9 via activation of extracellular signal-related kinase signaling in human ovarian cancer cells. These data suggest that tumor-associated macrophages can affect human ovarian cancer metastasis via the CCL7/CCR3 axis.

scholarly journals CHROMOSOMAL BASIS OF THE MEROZYGOSITY IN A PARTIALLY DIPLOID MUTANT OF PNEUMOCOCCUS

Genetics ◽  
1975 ◽  
Vol 80 (4) ◽  
pp. 667-678
Author(s):  
Mary Lee S Ledbetter ◽  
Rollin D Hotchkiss
Keyword(s):  
High Efficiency ◽  
Point Mutations ◽  
Resistant Mutant ◽  
Chromosomal Marker ◽  
Structural Abnormality ◽  
C Cells ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Low Efficiency ◽  
Derivatives Of

ABSTRACT A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

scholarly journals The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Anurag Kumar Sinha ◽  
Kristoffer Skovbo Winther
Keyword(s):  
Active Sites ◽  
Molecular Switch ◽  
Cell Physiology ◽  
Synthetase Activity ◽  
Wild Type ◽  
Functional Studies ◽  
Loop Region ◽  
A Site ◽  
Trna Binding

AbstractBacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity remain elusive. Here, we show a role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using mutagenesis screening and functional studies, we identify a loop region (residues 114–130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wild type RelA. Our data support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.

scholarly journals A procedure to introduce point mutations into the Rubisco large subunit gene in wild‐type plants

2021 ◽  
Author(s):  
Myat T. Lin ◽  
Douglas J. Orr ◽  
Dawn Worrall ◽  
Martin A. J. Parry ◽  
Elizabete Carmo‐Silva ◽  
...  
Keyword(s):  
Large Subunit ◽  
Point Mutations ◽  
Subunit Gene ◽  
Wild Type

scholarly journals Potential Anticancer Lipoxygenase Inhibitors from the Red Sea-Derived Brown Algae Sargassum cinereum: An In-Silico-Supported In-Vitro Study

Antibiotics ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 416
Author(s):  
Sami I. Alzarea ◽  
Abeer H. Elmaidomy ◽  
Hani Saber ◽  
Arafa Musa ◽  
Mohammad M. Al-Sanea ◽  
...  
Keyword(s):  
Red Sea ◽  
Antiproliferative Activity ◽  
Inhibitory Activity ◽  
Brown Algae ◽  
In Silico ◽  
Active Sites ◽  
In Vitro Study ◽  
Lipoxygenase Inhibitors ◽  
Phytochemical Investigation

LC-MS-assisted metabolomic profiling of the Red Sea-derived brown algae Sargassum cinereum “Sargassaceae” dereplicated eleven compounds 1–11. Further phytochemical investigation afforded two new aryl cresol 12–13, along with eight known compounds 14–21. Both new metabolites, along with 19, showed moderate in vitro antiproliferative activity against HepG2, MCF-7, and Caco-2. Pharmacophore-based virtual screening suggested both 5-LOX and 15-LOX as the most probable target linked to their observed antiproliferative activity. The in vitro enzyme assays revealed 12 and 13 were able to inhibit 5-LOX more preferentially than 15-LOX, while 19 showed a convergent inhibitory activity toward both enzymes. Further in-depth in silico investigation revealed the molecular interactions inside both enzymes’ active sites and explained the varying inhibitory activity for 12 and 13 toward 5-LOX and 15-LOX.

Sign in / Sign up

Export Citation Format

Share Document