Structural and functional characterization of the RBBP4–ZNF827 interaction and its role in NuRD recruitment to telomeres

2018 ◽  
Vol 475 (16) ◽  
pp. 2667-2679 ◽  
Author(s):  
Sile F. Yang ◽  
Ai-ai Sun ◽  
Yunyu Shi ◽  
Fudong Li ◽  
Hilda A. Pickett
Keyword(s):  
Amino Acids ◽  
Electrostatic Interactions ◽  
Zinc Finger Protein ◽  
Functional Characterization ◽  
Vital Role ◽  
Telomere Maintenance ◽  
Nurd Complex ◽  
Nucleosome Remodeling ◽  
Alternative Lengthening

The nucleosome remodeling and histone deacetylase (NuRD) complex is an essential multi-subunit protein complex that regulates higher-order chromatin structure. Cancers that use the alternative lengthening of telomere (ALT) pathway of telomere maintenance recruit NuRD to their telomeres. This interaction is mediated by the N-terminal domain of the zinc-finger protein ZNF827. NuRD–ZNF827 plays a vital role in the ALT pathway by creating a molecular platform for recombination-mediated repair. Disruption of NuRD binding results in loss of ALT cell viability. Here, we present the crystal structure of the NuRD subunit RBBP4 bound to the N-terminal 14 amino acids of ZNF827. RBBP4 forms a negatively charged channel that binds to ZNF827 through a network of electrostatic interactions. We identify the precise amino acids in RBBP4 required for this interaction and demonstrate that disruption of these residues prevents RBBP4 binding to both ZNF827 and telomeres, but is insufficient to decrease ALT activity. These data provide insights into the structural and functional determinants of NuRD activity at ALT telomeres.

scholarly journals STEM-13. FUNCTIONAL CHARACTERIZATION OF THE ZFHX4-CHD4 INTERACTION IN GLIOBLASTOMA CANCER STEM CELLS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii199-ii199
Author(s):  
Luciano Galdieri ◽  
Mitchell Grinwald ◽  
Zibi Gugala ◽  
Edward Oates ◽  
Milan Chheda
Keyword(s):  
Amino Acids ◽  
Stem Cells ◽  
Zinc Finger ◽  
Functional Characterization ◽  
Histone Deacetylation ◽  
Structural Basis ◽  
Nurd Complex ◽  
Nucleosome Remodeling ◽  
New Treatments ◽  
And Function

Abstract Glioblastoma stem cells (GSCs) they may be one reason for inevitable recurrence of GBM. We previously discovered that Zinc Finger Homeobox 4 (ZFHX4), a 450kD transcription factor, is required to maintain the GSC state. ZFHX4 interacts with CHD4, a core member of the nucleosome remodeling and deacetylase (NuRD) complex, which activates or represses gene expression via two distinct functions - histone deacetylation and ATP-dependent chromatin remodeling. CHD4 suppression phenocopies ZFHX4 suppression. The precise nature and function of the ZFHX4 interaction with CHD4 is not understood. Here we report that the ZFHX4-CHD4 interaction requires a single zinc-finger domain. An incremental truncation screen revealed that ZFHX4 amino acids 1838 to 2387, which contains zinc fingers 14 and 15, are required to bind CHD4. Disrupting the zinc coordination of zinc finger 14 impaired the ZFHX4-CHD4 interaction. Moreover, by overexpressing ZFHX4 amino acids 1838 to 2487, we decreased CHD4 recruitment to transcription regulatory regions of the stem cell genes SOX2 and SOX9, decreased transcription, and reduced clonogenic self-renewal. These results may provide the structural basis for new treatments to target GSCs and prevent recurrence in this devastating disease.

scholarly journals Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme

2006 ◽  
Vol 394 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Sandra Müller ◽  
Jennifer Disse ◽  
Manuela Schöttler ◽  
Sylvia Schön ◽  
Christian Prante ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Binding Capacity ◽  
Functional Characterization ◽  
Terminal Region ◽  
Binding Motif ◽  
Heparin Binding ◽  
Loss Of Function ◽  
The Impact

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

scholarly journals Functional characterization of constituent enzyme fractions of mycobacillin synthetase

1985 ◽  
Vol 230 (3) ◽  
pp. 785-789 ◽  
Author(s):  
S K Ghosh ◽  
S Majumder ◽  
N K Mukhopadhyay ◽  
S K Bose
Keyword(s):  
Amino Acids ◽  
Functional Characterization ◽  
Enzyme Fraction ◽  
Sequential Activation

The enzyme fraction A, a constituent enzyme of the three-fraction enzyme mycobacillin synthetase, independently and sequentially activated five amino acids starting from L-proline, producing the pentapeptide Pro(Asp1,Glu1,Tyr1)Asp. The fractions B and C were unable to function independently. However, the fraction B synthesized the nonapeptide Pro(Asp3,Glu1,Tyr2,Ser1)Leu, sequentially activating the pentapeptide and next four amino acids, whereas the fraction C synthesized mycobacillin by the sequential activation of the nonapeptide and the remaining four amino acids. The pH optima of the above enzymes are almost identical (pH 7.8), but their Km values are a little different.

Functional characterization of the transmembrane segment VII of the NHE1 isoform of the Na+/H+ exchangerThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

2007 ◽  
Vol 85 (3-4) ◽  
pp. 319-325 ◽  
Author(s):  
Jie Ding ◽  
Raymond W.P. Ng ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
70Th Birthday ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Transmembrane Segment ◽  
Transmembrane Segments ◽  
Pore Lining ◽  
Positively Charged ◽  
Selection Of

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH. It extrudes 1 intracellular H+ in exchange for 1 extracellular Na+. It has 2 large domains, an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the critical transmembrane segment TM VII. Residues Leu 255, Leu 258, Glu 262, Leu 265, Asn 266, Asp 267, Val 269, Val 272, and Leu 273 were all mutated to cysteine residues in the cysteineless NHE1 isoform. Mutation of amino acids E262, N266, and D267 caused severe defects in activity and targeting of the intact full length protein. The balance of the active mutants were examined for sensitivity to the sulfhydryl reactive reagents, positively charged MTSET ((2- (trimethylammonium)ethyl)methanethiosulfonate) and negatively charged MTSES ((2-sulfonatoethyl)methanethiosulfonate). Leu 255 and Leu 258 were sensitive to MTSET but not to MTSES. The results suggest that these amino acids are pore-lining residues. We present a model of TM VII that shows that residues Leu 255, Leu 258, Glu 262, Asn 266, and Asp 267 lie near the same face of TM VII, lining the ion transduction pore.

scholarly journals RsSOS1 Responding to Salt Stress Might Be Involved in Regulating Salt Tolerance by Maintaining Na+ Homeostasis in Radish (Raphanus sativus L.)

Horticulturae ◽  
2021 ◽  
Vol 7 (11) ◽  
pp. 458
Author(s):  
Wanting Zhang ◽  
Jingxue Li ◽  
Junhui Dong ◽  
Yan Wang ◽  
Liang Xu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Salt Stress ◽  
Salt Tolerance ◽  
Functional Characterization ◽  
Vital Role ◽  
Salt Stress Response ◽  
Reading Frame ◽  
Yield And Quality

Radish is a kind of moderately salt-sensitive vegetable. Salt stress seriously decreases the yield and quality of radish. The plasma membrane Na+/H+ antiporter protein Salt Overly Sensitive 1 (SOS1) plays a crucial role in protecting plant cells against salt stress, but the biological function of the RsSOS1 gene in radish remains to be elucidated. In this study, the RsSOS1 gene was isolated from radish genotype ‘NAU-TR17’, and contains an open reading frame of 3414 bp encoding 1137 amino acids. Phylogenetic analysis showed that RsSOS1 had a high homology with BnSOS1, and clustered together with Arabidopsis plasma membrane Na+/H+ antiporter (AtNHX7). The result of subcellular localization indicated that the RsSOS1 was localized in the plasma membrane. Furthermore, RsSOS1 was strongly induced in roots of radish under 150 mmol/L NaCl treatment, and its expression level in salt-tolerant genotypes was significantly higher than that in salt-sensitive ones. In addition, overexpression of RsSOS1 in Arabidopsis could significantly improve the salt tolerance of transgenic plants. Meanwhile, the transformation of RsSOS1△999 could rescue Na+ efflux function of AXT3 yeast. In summary, the plasma membrane Na+/H+ antiporter RsSOS1 plays a vital role in regulating salt-tolerance of radish by controlling Na+ homeostasis. These results provided useful information for further functional characterization of RsSOS1 and facilitate clarifying the molecular mechanism underlying salt stress response in radish.

Sign in / Sign up

Export Citation Format

Share Document