scholarly journals Design of genetically encoded sensors to detect nucleosome ubiquitination in live cells

2021 ◽  
Vol 220 (4) ◽  
Author(s):  
Carolina dos Santos Passos ◽  
Yun-Seok Choi ◽  
Christopher D. Snow ◽  
Tingting Yao ◽  
Robert E. Cohen
Keyword(s):  
Posttranslational Modifications ◽  
Fluorescent Proteins ◽  
Live Cells ◽  
Transcriptional Elongation ◽  
Binding Domains ◽  
Kd Values ◽  
Binding Peptides ◽  
Genetically Encoded Sensors ◽  
And Function ◽  
Nucleosome Binding

Histone posttranslational modifications (PTMs) are dynamic, context-dependent signals that modulate chromatin structure and function. Ubiquitin (Ub) conjugation to different lysines of histones H2A and H2B is used to regulate diverse processes such as gene silencing, transcriptional elongation, and DNA repair. Despite considerable progress made to elucidate the players and mechanisms involved in histone ubiquitination, there remains a lack of tools to monitor these PTMs, especially in live cells. To address this, we combined an avidity-based strategy with in silico approaches to design sensors for specifically ubiquitinated nucleosomes. By linking Ub-binding domains to nucleosome-binding peptides, we engineered proteins that target H2AK13/15Ub and H2BK120Ub with Kd values from 10−8 to 10−6 M; when fused to fluorescent proteins, they work as PTM sensors in cells. The H2AK13/15Ub-specific sensor, employed to monitor signaling from endogenous DNA damage through the cell cycle, identified and differentiated roles for 53BP1 and BARD1 as mediators of this histone PTM.

scholarly journals Spanning the gap: unraveling RSC dynamics in vivo

2021 ◽  
Author(s):  
Heinz Neumann ◽  
Bryan J. Wilkins
Keyword(s):  
Cell Cycle ◽  
Posttranslational Modifications ◽  
Transcriptional Activation ◽  
Binding Mode ◽  
Binding Modes ◽  
Binding Domains ◽  
Binding Interface ◽  
The Many ◽  
And Function

AbstractMultiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

scholarly journals The nucleosome acidic patch and H2A ubiquitination underlie mSWI/SNF recruitment in synovial sarcoma

2020 ◽  
Vol 27 (9) ◽  
pp. 836-845 ◽  
Author(s):  
Matthew J. McBride ◽  
Nazar Mashtalir ◽  
Evan B. Winter ◽  
Hai T. Dao ◽  
Martin Filipovski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Synovial Sarcoma ◽  
Direct Interaction ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Selective Targeting ◽  
Associated Proteins ◽  
And Function ◽  
Genomic Regions ◽  
Nucleosome Binding

AbstractInteractions between chromatin-associated proteins and the histone landscape play major roles in dictating genome topology and gene expression. Cancer-specific fusion oncoproteins, which display unique chromatin localization patterns, often lack classical DNA-binding domains, presenting challenges in identifying mechanisms governing their site-specific chromatin targeting and function. Here we identify a minimal region of the human SS18-SSX fusion oncoprotein (the hallmark driver of synovial sarcoma) that mediates a direct interaction between the mSWI/SNF complex and the nucleosome acidic patch. This binding results in altered mSWI/SNF composition and nucleosome engagement, driving cancer-specific mSWI/SNF complex targeting and gene expression. Furthermore, the C-terminal region of SSX confers preferential affinity to repressed, H2AK119Ub-marked nucleosomes, underlying the selective targeting to polycomb-marked genomic regions and synovial sarcoma–specific dependency on PRC1 function. Together, our results describe a functional interplay between a key nucleosome binding hub and a histone modification that underlies the disease-specific recruitment of a major chromatin remodeling complex.

scholarly journals Site-Specific Modification and Segmental Isotope Labelling of HMGN1 Reveals Long-Range Conformational Perturbations Caused by Posttranslational Modifications

2020 ◽  
Author(s):  
Gerhard Niederacher ◽  
Debra Urwin ◽  
David J. Tremethick ◽  
K. Johan Rosengren ◽  
Christian F. W. Becker ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
High Mobility ◽  
Intrinsically Disordered Protein ◽  
Site Specific ◽  
Isotope Labelling ◽  
Specific Modification ◽  
Intrinsically Disordered ◽  
Cellular Localisation ◽  
And Function ◽  
Nucleosome Binding

Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) - acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- and C-termini with segmental isotope labelling for NMR spectroscopy. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the roles of PTMs with atomic precision, from both structural and functional perspectives.<br>

scholarly journals Site-Specific Modification and Segmental Isotope Labelling of HMGN1 Reveals Long-Range Conformational Perturbations Caused by Posttranslational Modifications

2020 ◽  
Author(s):  
Gerhard Niederacher ◽  
Debra Urwin ◽  
David J. Tremethick ◽  
K. Johan Rosengren ◽  
Christian F. W. Becker ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
High Mobility ◽  
Intrinsically Disordered Protein ◽  
Site Specific ◽  
Isotope Labelling ◽  
Specific Modification ◽  
Intrinsically Disordered ◽  
Cellular Localisation ◽  
And Function ◽  
Nucleosome Binding

Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) - acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- and C-termini with segmental isotope labelling for NMR spectroscopy. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the roles of PTMs with atomic precision, from both structural and functional perspectives.<br>

scholarly journals Snails In Silico: A Review of Computational Studies on the Conopeptides

Marine Drugs ◽  
2019 ◽  
Vol 17 (3) ◽  
pp. 145 ◽  
Author(s):  
Rachael Mansbach ◽  
Timothy Travers ◽  
Benjamin McMahon ◽  
Jeanne Fair ◽  
S. Gnanakaran
Keyword(s):  
Posttranslational Modifications ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Defense Mechanism ◽  
Docking Studies ◽  
Chemical Diversity ◽  
Machine Learning Techniques ◽  
Peptide Toxins ◽  
Dynamics Simulations ◽  
And Function

Marine cone snails are carnivorous gastropods that use peptide toxins called conopeptides both as a defense mechanism and as a means to immobilize and kill their prey. These peptide toxins exhibit a large chemical diversity that enables exquisite specificity and potency for target receptor proteins. This diversity arises in terms of variations both in amino acid sequence and length, and in posttranslational modifications, particularly the formation of multiple disulfide linkages. Most of the functionally characterized conopeptides target ion channels of animal nervous systems, which has led to research on their therapeutic applications. Many facets of the underlying molecular mechanisms responsible for the specificity and virulence of conopeptides, however, remain poorly understood. In this review, we will explore the chemical diversity of conopeptides from a computational perspective. First, we discuss current approaches used for classifying conopeptides. Next, we review different computational strategies that have been applied to understanding and predicting their structure and function, from machine learning techniques for predictive classification to docking studies and molecular dynamics simulations for molecular-level understanding. We then review recent novel computational approaches for rapid high-throughput screening and chemical design of conopeptides for particular applications. We close with an assessment of the state of the field, emphasizing important questions for future lines of inquiry.

scholarly journals Structure and Function of the CFTR Chloride Channel

1999 ◽  
Vol 79 (1) ◽  
pp. S23-S45 ◽  
Author(s):  
DAVID N. SHEPPARD ◽  
MICHAEL J. WELSH
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Atp Hydrolysis ◽  
Current Knowledge ◽  
Structure And Function ◽  
Binding Domains ◽  
Transporter Family ◽  
Membrane Spanning ◽  
And Function ◽  
R Domain

Sheppard, David N., and Michael J. Welsh. Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79 , Suppl.: S23–S45, 1999. — The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl− channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.

ChemInform Abstract: The Structure and Function of Fluorescent Proteins

ChemInform ◽  
2009 ◽  
Vol 40 (50) ◽  
Author(s):  
Vedangi Sample ◽  
Robert H. Newman ◽  
Jin Zhang
Keyword(s):  
Fluorescent Proteins ◽  
Structure And Function ◽  
And Function

scholarly journals GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET

2009 ◽  
Vol 134 (6) ◽  
pp. 489-521 ◽  
Author(s):  
Fraser J. Moss ◽  
P.I. Imoukhuede ◽  
Kimberly Scott ◽  
Jia Hu ◽  
Joanna L. Jankowsky ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Fluorescent Proteins ◽  
Resonance Energy ◽  
High Order ◽  
Live Cells ◽  
Gaba Uptake ◽  
Cell Periphery ◽  
Wild Type ◽  
Gaba Transporter ◽  
Amplitude Component

The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ∼30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

scholarly journals Postprenylation CAAX Processing Is Required for Proper Localization of Ras but Not Rho GTPases

2005 ◽  
Vol 16 (4) ◽  
pp. 1606-1616 ◽  
Author(s):  
David Michaelson ◽  
Wasif Ali ◽  
Vi K. Chiu ◽  
Martin Bergo ◽  
Joseph Silletti ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Protein Trafficking ◽  
Rho Gtpases ◽  
Ras Proteins ◽  
Rho Proteins ◽  
Carboxyl Methylation ◽  
C Terminus ◽  
Individual Contributions ◽  
The Individual ◽  
And Function

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal– AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the –AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.

Identification of two independent nucleosome-binding domains in the transcriptional co-activator SPBP

2012 ◽  
Vol 442 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Sagar Darvekar ◽  
Sylvia Sagen Johnsen ◽  
Agnete Bratsberg Eriksen ◽  
Terje Johansen ◽  
Eva Sjøttem
Keyword(s):  
Transcription Factors ◽  
Dependent Manner ◽  
Chromatin Binding ◽  
Binding Region ◽  
Interaction Domain ◽  
Binding Domains ◽  
Element Binding Protein ◽  
Inducible Protein ◽  
Responsive Element ◽  
Nucleosome Binding

Transcriptional regulation requires co-ordinated action of transcription factors, co-activator complexes and general transcription factors to access specific loci in the dense chromatin structure. In the present study we demonstrate that the transcriptional co-regulator SPBP [stromelysin-1 PDGF (platelet-derived growth factor)-responsive element binding protein] contains two independent chromatin-binding domains, the SPBP-(1551–1666) region and the C-terminal extended PHD [ePHD/ADD (extended plant homeodomain/ATRX-DNMT3-DNMT3L)] domain. The region 1551–1666 is a novel core nucleosome-interaction domain located adjacent to the AT-hook motif in the DNA-binding domain. This novel nucleosome-binding region is critically important for proper localization of SPBP in the cell nucleus. The ePHD/ADD domain associates with nucleosomes in a histone tail-dependent manner, and has significant impact on the dynamic interaction between SPBP and chromatin. Furthermore, SPBP and its homologue RAI1 (retinoic-acid-inducible protein 1), are strongly enriched on chromatin in interphase HeLa cells, and both proteins display low nuclear mobility. RAI1 contains a region with homology to the novel nucleosome-binding region SPBP-(1551–1666) and an ePHD/ADD domain with ability to bind nucleosomes. These results indicate that the transcriptional co-regulator SPBP and its homologue RAI1 implicated in Smith–Magenis syndrome and Potocki–Lupski syndrome both belong to the expanding family of chromatin-binding proteins containing several domains involved in specific chromatin interactions.

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