Generation and functional characterization of a BCL10-inhibitory peptide that represses NF-κB activation

2009 ◽  
Vol 422 (3) ◽  
pp. 553-561 ◽  
Author(s):  
Daniela Marasco ◽  
Romania Stilo ◽  
Annamaria Sandomenico ◽  
Simona Maria Monti ◽  
Barbara Tizzano ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Nuclear Factor Κb ◽  
Lymphoid Cells ◽  
Amino Acid Residues ◽  
Inhibitory Peptide ◽  
Inhibitory Peptides ◽  
Cell Assays ◽  
Self Association ◽  
Competition Assays

The molecular complex containing BCL10 and CARMA [CARD (caspase recruitment domain)-containing MAGUK (membrane-associated guanylate kinase)] proteins has recently been identified as a key component in the signal transduction pathways that regulate activation of the transcription factor NF-κB (nuclear factor κB) in lymphoid and non-lymphoid cells. Assembly of complexes containing BCL10 and CARMA proteins relies on homophilic interactions established between the CARDs of these proteins. In order to identify BCL10-inhibitory peptides, we have established a method of assaying peptides derived from the CARD of BCL10 in binding competition assays of CARD–CARD self-association. By this procedure, a short peptide corresponding to amino acid residues 91–98 of BCL10 has been selected as an effective inhibitor of protein self-association. When tested in cell assays for its capacity to block NF-κB activation, this peptide represses activation of NF-κB mediated by BCL10, CARMA3 and PMA/ionomycin stimulation. Collectively, these results indicate that residues 91–98 of BCL10 are involved in BCL10 self-association and also participate in the interaction with external partners. We also show that blocking of the CARD of BCL10 may potentially be used for the treatment of pathological conditions associated with inappropriate NF-κB activation.

scholarly journals Characterization of COMMD protein–protein interactions in NF-κB signalling

2006 ◽  
Vol 398 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Prim de Bie ◽  
Bart van de Sluis ◽  
Ezra Burstein ◽  
Karen J. Duran ◽  
Ruud Berger ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Copper Metabolism ◽  
Nuclear Factor Κb ◽  
Inhibitory Effect ◽  
Protein Family ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Necrosis Factor

COMMD [copper metabolism gene MURR1 (mouse U2af1-rs1 region 1) domain] proteins constitute a recently identified family of NF-κB (nuclear factor κB)-inhibiting proteins, characterized by the presence of the COMM domain. In the present paper, we report detailed investigation of the role of this protein family, and specifically the role of the COMM domain, in NF-κB signalling through characterization of protein–protein interactions involving COMMD proteins. The small ubiquitously expressed COMMD6 consists primarily of the COMM domain. Therefore COMMD1 and COMMD6 were analysed further as prototype members of the COMMD protein family. Using specific antisera, interaction between endogenous COMMD1 and COMMD6 is described. This interaction was verified by independent techniques, appeared to be direct and could be detected throughout the whole cell, including the nucleus. Both proteins inhibit TNF (tumour necrosis factor)-induced NF-κB activation in a non-synergistic manner. Mutation of the amino acid residues Trp24 and Pro41 in the COMM domain of COMMD6 completely abolished the inhibitory effect of COMMD6 on TNF-induced NF-κB activation, but this was not accompanied by loss of interaction with COMMD1, COMMD6 or the NF-κB subunit RelA. In contrast with COMMD1, COMMD6 does not bind to IκBα (inhibitory κBα), indicating that both proteins inhibit NF-κB in an overlapping, but not completely similar, manner. Taken together, these data support the significance of COMMD protein–protein interactions and provide new mechanistic insight into the function of this protein family in NF-κB signalling.

scholarly journals Functional characterization of human osteoclast inhibitory peptide-1 (OIP-1/hSca) gene promoter

Gene ◽  
2006 ◽  
Vol 371 (1) ◽  
pp. 16-24 ◽  
Author(s):  
Shanmugarajan Srinivasan ◽  
Masahiro Ito ◽  
Hiroshi Kajiya ◽  
L. Lyndon Key ◽  
Theresa L. Johnson-Pais ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Gene Promoter ◽  
Inhibitory Peptide ◽  
Human Osteoclast

Prokaryotic Expression and Functional Characterization of the 19 kDa Protein in Balanus albicostatus Cement

2013 ◽  
Vol 461 ◽  
pp. 445-450
Author(s):  
Chao Liang ◽  
Yun Qiu Li ◽  
Bi Ru Hu ◽  
Wen Jian Wu
Keyword(s):  
Amino Acid ◽  
Molecular Design ◽  
Functional Characterization ◽  
Blue Staining ◽  
Amino Acid Residues ◽  
Adhesive Ability ◽  
Solid Liquid ◽  
Dental Applications ◽  
The Relationship

Barnacle is a unique sessile crustacean, which produces a multi-protein complex historically called barnacle cement to attach to diverse immersed materials permanently. The proteinaceous cement exhibits powerful adhesive property and special waterproof capability to cure at solid-liquid boundaries, which makes it ideal biomaterial for technical, medical and dental applications. It has been proved that a 19 kDa protein component, termed cp-19k in the cement plays a key role in surface coupling during underwater attachment. To verify whether the bacterial recombinant 19 kDa protein retains the adhesive ability, we cloned and sequenced the Bacp-19k gene in Balanus albicostatus. It encodes 173 amino acid residues, with seven biased ones, Thr, Lys, Gly, Ala, Val, Ser and Leu, comprising about 80% of the total. Two amino acid substitutions (F69L, I106L) were discovered in Bacp-19k due to the polymorphisms in barnacle cp-19ks, compared with the submitted one (GenBank: AB242295.1). Recombinant Bacp-19k was highly expressed in host strain Escherichia coli BL21 (DE3) and purified by affinity chromatography. Adsorption of recombinant Bacp-19k to glass substrata was examined by Coomassie brilliant blue staining. Future study will reveal the relationship between specific structures and functions for molecular design of novel biomimetic underwater adhesives.

scholarly journals B-lymphocyte heterogeneity: development and characterization of an alloantiserum which distinguishes B-lymphocyte differentiation alloantigens.

1977 ◽  
Vol 145 (1) ◽  
pp. 101-110 ◽  
Author(s):  
A Ahmed ◽  
I Scher ◽  
S O Sharrow ◽  
A H Smith ◽  
W E Paul ◽  
...  
Keyword(s):  
B Lymphocytes ◽  
B Lymphocyte ◽  
Functional Characterization ◽  
High Ratio ◽  
Lymphoid Cells ◽  
Male Mice ◽  
Spleen Cells ◽  
Immune Defect ◽  
Intermediate Density

CBA/N mice and F1 male mice, which are hemizygous for the CBA/N X chromosome, have an immune defect which is associated with the absence (deficiency) of a subpopulation of mature or late developing B lymphocytes. This characteristic was utilized to develop an antiserum that was specific for this subclass of B cells. C57BL/L mice were immunized with DBA/2 spleen calls, and the resulting antisera was absorbed with lymphoid cells derived from immunologically abnormal (CBA/N female X DBA/2 male)F1 male mice. The absorbed antisera was cytotoxic for a subpopulation of lymphocytes that was present in the spleens of adult DBA/2 and (CBA/N female X DBA/2 male)F1 female mice. The cells killed by the absorbed antisera were Ig-bearing, complement receptor-bearing B lymphocytes, which had a low-to-intermediate density of total surface Ig. Moreover, the cells remaining after treatment of adult (CBA/N female X DBA/2 male)F1 female spleen cells with the absorbed antisera and C had a high ratio of surface IgM to IgD. The development of this cytotoxic alloantisera, which is specific for a late developing (mature) subpopulation of B lymphocytes, will allow the functional characterization of subclasses of B lymphocytes.

scholarly journals Yeast Cell-Based Transport Assay for the Functional Characterization of Human 4F2hc-LAT1 and ‐LAT2, and LAT1 and LAT2 Substrates and Inhibitors

2021 ◽  
Vol 8 ◽  
Author(s):  
Satish Kantipudi ◽  
Dimitrios Fotiadis
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Functional Characterization ◽  
Methylotrophic Yeast ◽  
Cell Types ◽  
Amino Acid Transporters ◽  
Cell Assays ◽  
Transport Assay ◽  
Compound Screening

In mammalian cells, the L-type amino acid transporters (LATs) LAT1 (SLC7A5) and LAT2 (SLC7A8) form heterodimeric amino acid transporters (HATs) with the ancillary protein 4F2hc and are involved in the cellular uptake of specific amino acids. The HAT 4F2hc-LAT1 is found upregulated in various cancer cell types, while 4F2hc-LAT2 is a transporter for non-cancer cells. Preclinical studies have highlighted that 4F2hc-LAT1 plays an important role in tumor progression representing a valid anticancer target. Consequently, current research is focusing on the development of potent and specific human 4F2hc-LAT1 inhibitors. On the other hand, 4F2hc-LAT2 is emerging as target of other diseases, thus also gaining clinical interest. To determine affinity and specificity of substrates and inhibitors for 4F2hc-LAT1 or 4F2hc-LAT2, robust transport cell assays are indispensable. We have optimized and validated a transport assay using cells of the methylotrophic yeast Pichia pastoris stably overexpressing the human HATs 4F2hc-LAT1 or -LAT2, and the LATs LAT1 or LAT2 alone. The radioligand [3H]L-leucine was used as reporter and the substrates L-leucine, triiodothyronine (T3) and thyroxine (T4) as well as the inhibitors BCH and JPH203 (KYT-0353) for assay validation. Obtained half-maximal inhibitory concentrations also provided new insights, e.g., into the LAT specificity of the potent inhibitor JPH203 and on the potency of the thyroid hormones T3 and T4 to inhibit transport through human 4F2hc-LAT2. The LAT1 and LAT2 assays are of particular interest to determine possible implications and influences of 4F2hc in ligand binding and transport. In summary, the presented assays are valuable for characterization of ligands, e.g., towards 4F2hc-LAT1 specificity, and can also be applied for compound screening. Finally, our established approach and assay would also be applicable to other HATs and LATs of interest.

scholarly journals Structural and functional characterization of SplA, an exclusively specific protease of Staphylococcus aureus

2009 ◽  
Vol 419 (3) ◽  
pp. 555-564 ◽  
Author(s):  
Justyna Stec-Niemczyk ◽  
Katarzyna Pustelny ◽  
Magdalena Kisielewska ◽  
Michal Bista ◽  
Kevin T. Boulware ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Expression System ◽  
Functional Characterization ◽  
Physiological Role ◽  
Peptide Bond ◽  
Amino Acid Residues ◽  
Specific Protease

Staphylococcus aureus is a dangerous human pathogen whose antibiotic resistance is steadily increasing and no efficient vaccine is as yet available. This serious threat drives extensive studies on staphylococcal physiology and pathogenicity pathways, especially virulence factors. Spl (serine protease-like) proteins encoded by an operon containing up to six genes are a good example of poorly characterized secreted proteins probably involved in virulence. In the present study, we describe an efficient heterologous expression system for SplA and detailed biochemical and structural characterization of the recombinant SplA protease. The enzyme shares a significant sequence homology to V8 protease and epidermolytic toxins which are well documented staphylococcal virulence factors. SplA has a very narrow substrate specificity apparently imposed by the precise recognition of three amino acid residues positioned N-terminal to the hydrolysed peptide bond. To explain determinants of this extended specificity we resolve the crystal structure of SplA and define the consensus model of substrate binding. Furthermore we demonstrate that artificial N-terminal elongation of mature SplA mimicking a naturally present signal peptide abolishes enzymatic activity. The probable physiological role of the process is discussed. Of interest, even though precise N-terminal trimming is a common regulatory mechanism among S1 family enzymes, the crystal structure of SplA reveals novel significantly different mechanistic details.

Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine

2004 ◽  
Vol 287 (1) ◽  
pp. G236-G242 ◽  
Author(s):  
Qiang Li ◽  
Andrei Manolescu ◽  
Mabel Ritzel ◽  
Sylvia Yao ◽  
Melissa Slugoski ◽  
...  
Keyword(s):  
Small Intestine ◽  
Brush Border Membrane ◽  
Functional Characterization ◽  
Northern Blotting ◽  
Rat Tissues ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Structural Determinants ◽  
Close Sequence

Facilitated glucose transporters (GLUTs) mediate transport of sugars across cell membranes by using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this work a novel hexose transporter isoform, GLUT7, has been cloned from a human intestinal cDNA library by using a PCR-based strategy (GenBank accession no. AY571960 ). The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes, it showed high-affinity transport for glucose ( Km = 0.3 mM) and fructose (IC50 = 0.060 mM). Galactose, 2-deoxy-d-glucose, and xylose were not transported. Uptake of 100 μM d-glucose was not inhibited by 200 μM phloretin or 100 μM cytochalasin B. Northern blotting indicated that the mRNA for GLUT7 is present in the human small intestine, colon, testis, and prostate. Western blotting and immunohistochemistry of rat tissues with an antibody raised against the predicted COOH-terminal sequence confirmed expression of the protein in the small intestine and indicated that the transporter is predominantly expressed in the enterocytes' brush-border membrane. The unusual substrate specificity and close sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUTs and the class I member GLUT2. Comparison between these proteins may provide key information as to the structural determinants for the recognition of fructose as a substrate.

scholarly journals Functional Characterization of a Trehalose-6-Phosphate Synthase in Diaphorina citri Revealed by RNA Interference and Transcriptome Sequencing

Insects ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1074
Author(s):  
Jian-Chun Song ◽  
Zhan-Jun Lu ◽  
Long Yi ◽  
Hai-Zhong Yu
Keyword(s):  
Fatty Acid ◽  
Rna Interference ◽  
Functional Characterization ◽  
Instar Nymph ◽  
Amino Acid Residues ◽  
Diaphorina Citri ◽  
Real Time Quantitative Pcr ◽  
Qpcr Analysis ◽  
Phosphate Synthase

Trehalose-6-phosphate synthase (TPS) plays an important role in the synthesis of trehalose. In the current study, a TPS gene was obtained from Diaphorina citri, and named as DcTPS1 which encoded a protein of 833 amino acid residues. Real-time quantitative PCR (qPCR) analysis revealed that DcTPS1 had the highest expression level in the midgut and fifth-instar nymph stage. Knockdown of DcTPS1 by RNA interference (RNAi) induced an abnormal phenotype and increased mortality and malformation rate with a decreased molting rate. In addition, silencing of DcTPS1 significantly inhibited D. citri chitin metabolism and fatty acid metabolism, while the expression levels of fatty acid decomposition-related genes were downregulated. Furthermore, comparative transcriptomics analysis revealed that 791 differentially expressed genes (DEGs) were upregulated and 678 DEGs were downregulated when comparing dsDcTPS1 groups with dsGFP groups. Bioinformatics analysis showed that upregulated DEGs were mainly involved in oxidative phosphorylation, whereas downregulated DEGs were mainly attributed to the lysosome and ribosome. These results indicated that DcTPS1 played an important role in the growth and development of D. citri.

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