scholarly journals Integrative Modeling of a Sin3/HDAC Complex Sub-structure

2019 ◽  
Author(s):  
Charles A. S. Banks ◽  
Ying Zhang ◽  
Sayem Miah ◽  
Yan Hao ◽  
Mark K. Adams ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Spatial Arrangement ◽  
Scaffolding Protein ◽  
Cross Link ◽  
High Confidence ◽  
Affinity Capture ◽  
Existing Structures ◽  
Distance Restraints ◽  
Cross Links ◽  
Sin3 Complex

AbstractSin3/HDAC complexes function by deacetylating histones, which makes chromatin more compact and modulates gene expression. Although components used to build these complexes have been well defined, we still have only a limited understanding of the structure of the Sin3/HDAC subunits as they are assembled around the scaffolding protein SIN3A. To characterize the spatial arrangement of Sin3 subunits, we combined Halo affinity capture, chemical cross-linking and high-resolution mass spectrometry (XL-MS) to determine intersubunit distance constraints, identifying 66 high-confidence interprotein and 63 high-confidence self cross-links for 13 Sin3 subunits. To validate our XL-MS data, we first mapped self cross-links onto existing structures to verify that cross-link distances were consistent with cross-linker length and subsequently deleted crosslink hotspot regions within the SIN3A scaffolding protein which then failed to capture crosslinked partners. Having assessed cross-link authenticity, we next used distance restraints from interprotein cross-links to guide assembly of a Sin3 complex substructure. We identified the relative positions of subunits SAP30L, HDAC1, SUDS3, HDAC2, and ING1 around the SIN3A scaffold. The architecture of this subassembly suggests that multiple factors have space to assemble to collectively influence the behavior of the catalytic subunit HDAC1.

scholarly journals Analysis of the impact of molecular motions on the efficiency of XL-MS and the distance restraints in hybrid structural biology

2018 ◽  
Author(s):  
Vladimir Svetlov ◽  
Evgeny Nudler
Keyword(s):  
Structural Biology ◽  
Protein Complexes ◽  
Protein Docking ◽  
Protein Fold ◽  
Molecular Motions ◽  
Cross Link ◽  
Distance Restraints ◽  
Cross Links ◽  
Cross Linker ◽  
The Impact

AbstractCovalent cross-link mapping by mass spectrometry (XL-MS) is rapidly becoming the most widely used method of hybrid structural biology. We investigated the impact of incremental variations of cross-linker length have on the depth of XL-MS interrogation of protein-protein complexes, and assessed the role molecular motions in solution play in generation of cross-link-derived distance restraints. Supplementation of a popular NHS-ester cross-linker, DSS, with 2 reagents shorter or longer by CH2-CH2, increased the number of non-reductant cross-links by ~50%. Molecular dynamics simulations of these cross-linkers revealed 3 individual, partially overlapping ranges of motion, consistent with partially overlapping sets of cross-links formed by each reagent. Similar simulations elucidated protein fold-specific ranges of motions for the reactive and backbone atoms from rigid and flexible target domains. Together these findings create a quantitative framework for generation of cross-linker- and protein fold-specific distance restraints for XL-MS-guided protein-protein docking.

scholarly journals Chemistry of the collagen cross-links. Isolation and characterization of two intermediate intermolecular cross-links in collagen

1970 ◽  
Vol 117 (5) ◽  
pp. 819-831 ◽  
Author(s):  
A. J. Bailey ◽  
Catherine M. Peach ◽  
L. J. Fowler
Keyword(s):  
Mass Spectrometry ◽  
Large Scale ◽  
High Resolution Mass Spectrometry ◽  
Collagen Fibre ◽  
Reduced Form ◽  
Cross Link ◽  
Isolation And Characterization ◽  
Cross Links

This paper describes the isolation from reduced collagen of two new amino acids believed to be involved, in their non-reduced form, as intermolecular cross-links stabilizing the collagen fibre. The reduction of intact collagen fibrils with tritiated sodium borohydride was found to stabilize the aldehyde-mediated cross-links to acid hydrolysis and thus allowed their location and isolation from acid hydrolysates on an automatic amino acid analyser. Comparison of the radioactive elution patterns from the autoanalyser of collagen treated in various ways before reduction permitted a preliminary classification of the peaks into cross-link precursors, intramolecular and intermolecular cross-links. The techniques employed to isolate the purified components on a large scale and to identify them structurally are described in detail. Two labile intermolecular cross-links were isolated in their reduced forms, one of which was identified by high-resolution mass spectrometry as N∈-(5-amino-5-carboxypentyl)hydroxylysine. The structure of this compound was confirmed by chemical synthesis. The cross-link precursor α-aminoadipic δ-semialdehyde was isolated in its reduced form, ∈-hydroxynorleucine, together with its acid degradation product ∈-chloronorleucine. A relatively stable intermolecular cross-link was isolated and partially characterized by mass spectrometry as an aldol resulting from the reaction of the δ-semialdehyde derived from lysine and hydroxylysine.

scholarly journals Linear Dynamic Viscoelasticity of Dual Cross-Link Poly(Vinyl Alcohol) Hydrogel with Determined Borate Ion Concentration

Gels ◽  
2021 ◽  
Vol 7 (2) ◽  
pp. 71
Author(s):  
Takuro Taniguchi ◽  
Kenji Urayama
Keyword(s):  
Vinyl Alcohol ◽  
Ion Concentration ◽  
Poly Vinyl Alcohol ◽  
Relaxation Dynamics ◽  
Cross Link ◽  
Dynamic Viscoelasticity ◽  
Linear Dynamic ◽  
Cross Links ◽  
Pva Gel ◽  
Pva Hydrogel

We investigated the linear dynamic viscoelasticity of dual cross-link (DC) poly(vinyl alcohol) (PVA) (DC-PVA) hydrogels with permanent and transient cross-links. The concentrations of incorporated borate ions to form transient cross-links in the DC-PVA hydrogels (CBIN) were determined by the azomethine-H method. The dynamic viscoelasticity of the DC-PVA hydrogel cannot be described by a simple sum of the dynamic viscoelasticity of the PVA gel with the same permanent cross-link concentration and the PVA aqueous solution with the same borate ion concentration (CB = CBIN) as in the DC-PVA gel. The DC-PVA hydrogel exhibited a considerably higher relaxation strength, indicating that the introduction of permanent cross-links into temporary networks increases the number of viscoelastic chains with finite relaxation times. In contrast, the relaxation frequency (ωc) (given by the frequency at the maximum of loss modulus) for the DC-PVA hydrogel was slightly lower but comparable to that for a dilute PVA solution with the same CB. This signifies that the relaxation dynamics of the DC-PVA hydrogels is essentially governed by the lifetime of an interchain transient cross-link (di-diol complex of boron). The effect of permanent cross-linking on the relaxation dynamics was observed in the finite broadening of the relaxation-time distribution in the long time region.

scholarly journals Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro.

1986 ◽  
Vol 103 (1) ◽  
pp. 23-31 ◽  
Author(s):  
E J Aamodt ◽  
J G Culotti
Keyword(s):  
Caenorhabditis Elegans ◽  
Apparent Molecular Weight ◽  
Cross Link ◽  
Nematode Caenorhabditis Elegans ◽  
Microtubule Associated Proteins ◽  
C Elegans ◽  
Associated Proteins ◽  
Cross Links ◽  
The Cross

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.

EXPERIMENTAL VALIDATION OF THE CHARLESBY AND HORIKX MODELS APPLIED TO DE-VULCANIZATION OF SULFUR AND PEROXIDE VULCANIZATES OF NR AND EPDM

2016 ◽  
Vol 89 (4) ◽  
pp. 671-688 ◽  
Author(s):  
M. A. L. Verbruggen ◽  
L. van der Does ◽  
W. K. Dierkes ◽  
J. W. M. Noordermeer
Keyword(s):  
Experimental Validation ◽  
Main Chain ◽  
Sol Gel ◽  
Chain Scission ◽  
Cross Linking ◽  
Cross Link ◽  
Practical Reality ◽  
Cross Links ◽  
The Cross ◽  
Theoretical Considerations

ABSTRACT The theoretical model developed by Charlesby to quantify the balance between cross-links creation of polymers and chain scission during radiation cross-linking and further modifications by Horikx to describe network breakdown from aging were merged to characterize the balance of both types of scission on the development of the sol content during de-vulcanization of rubber networks. There are, however, disturbing factors in these theoretical considerations vis-à-vis practical reality. Sulfur- and peroxide-cured NR and EPDM vulcanizates were de-vulcanized under conditions of selective cross-link and random main-chain scissions. Cross-link scission was obtained using thiol-amine reagents for selective cleavage of sulfur cross-links. Random main-chain scission was achieved by heating peroxide vulcanizates of NR with diphenyldisulfide, a method commonly employed for NR reclaiming. An important factor in the analyses of these experiments is the cross-linking index. Its value must be calculated using the sol fraction of the cross-linked network before de-vulcanization to obtain reliable results. The values for the cross-linking index calculated with sol-gel data before de-vulcanization appear to fit the experimentally determined modes of network scission during de-vulcanization very well. This study confirms that the treatment of de-vulcanization data with the merged Charlesby and Horikx models can be used satisfactorily to characterize the de-vulcanization of NR and EPDM vulcanizates.

Role of the FAD-dependent polyamine oxidase in the selective formation of N1,N8-bis(γ- glutamyl)spermidine protein cross-links1

2007 ◽  
Vol 35 (2) ◽  
pp. 396-400 ◽  
Author(s):  
A. Lentini ◽  
P. Mattioli ◽  
B. Provenzano ◽  
A. Abbruzzese ◽  
M. Caraglia ◽  
...  
Keyword(s):  
Stratum Corneum ◽  
Polyamine Oxidase ◽  
Polyamine Metabolism ◽  
Suitable Model ◽  
Cross Link ◽  
Cellular Mechanisms ◽  
Preferential Formation ◽  
Cross Links ◽  
Selective Formation

Protein-bound γ-glutamylpolyamines have highlighted a new pathway in polyamine metabolism. Human foreskin keratinocytes offer a suitable model for this study. Indeed, they develop polymerized envelopes, as they differentiate, rich in ϵ-(γ-glutamyl)lysine and N1,N8-bis(γ-glutamyl)spermidine cross-links. We have found that the selective oxidation of N1-(γ-glutamyl)spermidine and N-(γ-glutamyl)spermine by FAD-dependent polyamine oxidase (PAO) may be one of the cellular mechanisms regulating the preferential formation of a sterically defined bis(γ-glutamyl)spermidine cross-link. The significance of this finding is unknown, but it suggests that the target of this PAO-modulation is to achieve the biochemical prerequisite for production of a normal epidermal stratum corneum.

scholarly journals Magnetic composites based on NR and strontium ferrite

2019 ◽  
Vol 12 (1) ◽  
pp. 63-69
Author(s):  
Ján Kruželák ◽  
Andrea Kvasničáková ◽  
Rastislav Dosoudil ◽  
Ivan Hudec
Keyword(s):  
Carbon Black ◽  
Strontium Ferrite ◽  
Rubber Matrix ◽  
Cross Link ◽  
Magnetic Composites ◽  
Link Density ◽  
Cross Links ◽  
Cross Link Density ◽  
The Cross ◽  
Thermo Physical Properties

Abstract Two types of composites based on natural rubber (NR) and strontium ferrite were tested in this study. Composites of the first type were prepared by incorporation of strontium ferrite in the concentration range ranging from 0 to 100 phr (parts per hundred rubber) into pure NR based rubber matrix, while with those of the second type, strontium ferrite was dosed in the same concentration level into NR based rubber batch with constant amount of carbon black — 25 phr. For rubber matrices cross-linking, a standard sulfur based curing system was used. This work is focused on the effect of magnetic filler content on physico-mechanical, magnetic and thermo-physical properties of composite materials. Subsequently, the cross-link density and the structure of the formed sulfidic cross-links were examined. The results showed that the cross-link density of both types of composites increased with the increasing content of magnetic filler, while the structure of the sulfidic cross-links was almost not influenced by the amount of strontium ferrite. Tensile strength of rubber composites with pure rubber matrix was slightly improved by the incorporation of ferrite, while in case of composites based on a carbon black batch, the incorporation of magnetic filler resulted in the decrease of this characteristic. The presence of magnetic filler in both types of composites leads to a significant increase of the remanent magnetic induction.

N,N′-Ethylene-bis(iodoacetamide) as a probe for structural and functional characteristics of brine shrimp, squid, and bovine tubulins

1985 ◽  
Vol 63 (6) ◽  
pp. 439-447 ◽  
Author(s):  
Richard F. Luduena ◽  
Mary Carmen Roach ◽  
Thomas H. MacRae ◽  
George M. Langford
Keyword(s):  
Electrophoretic Mobility ◽  
Brine Shrimp ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Cross Link ◽  
Unique Effect ◽  
Ligand Binding Sites ◽  
Cross Links ◽  
Sulfate Formation ◽  
Brain Tubulin

We have developed a simple probe for certain functionally significant features of the tubulin molecule. When bovine brain tubulin is treated with N,N′-ethylene-bis(iodoacetamide) (EBI), two intrachain cross-links, designated βs and β*, are formed in β-tubulin, each one with a unique effect on the electrophoretic mobility of β on gels containing sodium dodecyl sulfate. Formation of the β* cross-link, which involves at least one assembly-critical sulfhydryl, is completely inhibited by colchicine and its congeners, while that of βs is inhibited completely by maytansine and GTP and partly by vinblastine. To see how conserved this complex pattern is in evolution we examined tubulins from the brine shrimp Artemia and the squid Loligo. In both tubulins EBI forms the β* cross-link in a reaction inhibitable by colchicine, podophyllotoxin, and nocodazole. In each tubulin, EBI appears to form a second intrachain cross-link in a reaction that can be inhibited completely by maytansine and GTP and partly by vinblastine. In Artemia, this cross-link alters the electrophoretic mobility to a slightly smaller extent than is the case for βs in bovine brain, but in Loligo the alteration is much greater. It seems that the ligand-binding sites, the critical sulfhydryls, and their spatial interrelationships are strongly conserved and that the βs sulfhydryls or the sequence between them are less strongly conserved in evolution.

scholarly journals Two DD-Carboxypeptidases fromMycobacterium smegmatisAffect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Satya Deo Pandey ◽  
Shilpa Pal ◽  
Ganesh Kumar N ◽  
Ankita Bansal ◽  
Sathi Mallick ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Cross Linking ◽  
Surface Lipid ◽  
Cross Link ◽  
Content Type ◽  
Murine Macrophages ◽  
Cross Links ◽  
Link Formation

ABSTRACTDuring the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between twomeso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (betweend-Ala andmeso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. Thedd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used byld-transpeptidases as substrates to form 3-3 cross-links. Therefore,dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology ofdd-CPases in mycobacteria is relatively unexplored. In this study, we deleted twodd-CPase genes,msmeg_2433andmsmeg_2432, both individually and in combination, fromMycobacterium smegmatismc2155. Though the singledd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant,viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of thedd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCEThe glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. Thedd-CPases generate tetrapeptides by acting on the pentapeptides, andld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of twodd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, includingMycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology ofdd-CPases might help us design antimycobacterial therapeutics in the future.

Chemical and biochemical properties of abasic site adducts and 6-thioguanine DNA cross-links in DNA

2017 ◽  
Author(s):  
◽  
Calvin D. Lewis
Keyword(s):  
Excision Repair ◽  
Biochemical Properties ◽  
Macromolecular Complex ◽  
Abasic Site ◽  
Cross Link ◽  
Dna Lesion ◽  
Phosphate Backbone ◽  
Cross Links ◽  
Ap Sites ◽  
Cellular Dna

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] DNA is a macromolecular complex, composed of the nucleotides adenine, thymine, guanine and cytosine interconnected by a phosphate backbone, that contains the genetic code for living organisms and viruses. Spontaneous and enzymatic hydrolysis of the glycosidic bonds that hold the coding nucleobases to the 2-deoxyribose-phosphate backbone of DNA results in the production of abasic (Ap) sites. These lesions are abundant in cellular DNA, and cellular Ap-containing DNA is damaging and may lead to cellular destruction if left unrepaired. Thus, efficient cellular DNA repair mechanisms that repair Ap sites have evolved in DNA containing organisms. The studies in this report examine the interaction between small molecules or naturally occurring DNA residues with Ap sites in duplex DNA. Experiments provide evidence that hydralazine binds to and forms a stable DNA lesion in single- and double-stranded DNA. Also, the hydralazine-DNA lesion is found to be a poor substrate for mammalian base excision repair enzymes such as Ap endonuclease and 8-oxoguanine DNA glycosylase. In addition, these studies provide preliminary evidence that hydralazine may potentiate the cytotoxicity of temozolomide in U87 cells. The investigation of the formation of cross-links between canonical DNA residues deoxyadenosine (dA) and deoxyguanosine (dG) with Ap sites is also explored. These experiments suggest that sequence effects contribute majorly to the cross-link yield in both dA- and dG-Ap site cross-links, especially when comparing central versus terminal cross-link locations. Here, this manuscript provides novel studies involving the interaction between DNA analog 6-thioguanine and opposing DNA bases in duplex oligonucleotide DNA.

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