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1.
IUCrJ ; 11(Pt 5): 780-791, 2024 Sep 01.
Article in English | MEDLINE | ID: mdl-39008358

ABSTRACT

The advent of serial crystallography has rejuvenated and popularized room-temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g. large complexes, membrane proteins and viruses) form fragile crystals that are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallization. Many medically relevant samples are expressed in mammalian cell lines, producing a meager quantity of protein that is incompatible with batch crystallization. This can limit the scope of serial crystallography approaches. Direct in situ data collection from a 96-well crystallization plate enables not only the identification of the best diffracting crystallization condition but also the possibility for structure determination under ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallization plates mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility. We applied our iSX approach on a challenging project, autotaxin, a therapeutic target expressed in a stable human cell line, to determine the structure in the lowest-symmetry P1 space group at 3.0 Šresolution. Our in situ data collection strategy provided a complete dataset for structure determination while screening various crystallization conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallization plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons.


Subject(s)
Crystallization , Crystallography, X-Ray/methods , Humans , Protein Conformation , Synchrotrons
2.
Chembiochem ; 25(12): e202400235, 2024 Jun 17.
Article in English | MEDLINE | ID: mdl-38642076

ABSTRACT

The pigmentation of the skin, modulated by different actors in melanogenesis, is mainly due to the melanins (protective pigments). In humans, these pigments' precursors are synthetized by an enzyme known as tyrosinase (TyH). The regulation of the enzyme activity by specific modulators (inhibitors or activators) can offer a means to fight hypo- and hyper-pigmentations responsible for medical, psychological and societal handicaps. Herein, we report the investigation of phenylalanine derivatives as TyH modulators. Interacting with the binuclear copper active site of the enzyme, phenylalanine derivatives combine effects induced by combination with known resorcinol inhibitors and natural substrate/intermediate (amino acid part). Computational studies including docking, molecular dynamics and free energy calculations combined with biological activity assays on isolated TyH and in human melanoma MNT-1 cells, and X-ray crystallography analyses with the TyH analogue Tyrp1, provide conclusive evidence of the interactions of phenylalanine derivatives with human tyrosinase. In particular, our findings indicate that an analogue of L-DOPA, namely (S)-3-amino-tyrosine, stands out as an amino phenol derivative with inhibitory properties against TyH.


Subject(s)
Enzyme Inhibitors , Monophenol Monooxygenase , Phenylalanine , Humans , Monophenol Monooxygenase/metabolism , Monophenol Monooxygenase/antagonists & inhibitors , Monophenol Monooxygenase/chemistry , Phenylalanine/chemistry , Phenylalanine/metabolism , Phenylalanine/analogs & derivatives , Phenylalanine/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/chemical synthesis , Molecular Docking Simulation , Crystallography, X-Ray , Molecular Dynamics Simulation , Catalytic Domain , Molecular Structure
3.
Nat Commun ; 14(1): 8248, 2023 Dec 12.
Article in English | MEDLINE | ID: mdl-38086790

ABSTRACT

The Mitochondrial Complex I Assembly (MCIA) complex is essential for the biogenesis of respiratory Complex I (CI), the first enzyme in the respiratory chain, which has been linked to Alzheimer's disease (AD) pathogenesis. However, how MCIA facilitates CI assembly, and how it is linked with AD pathogenesis, is poorly understood. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, releasing the FAD cofactor and converting ACAD9 from a fatty acid ß-oxidation (FAO) enzyme to a CI assembly factor. We provide evidence that ECSIT phosphorylation downregulates its association with ACAD9 and is reduced in neuronal cells upon exposure to amyloid-ß (Aß) oligomers. These findings advance our understanding of the MCIA complex assembly and suggest a possible role for ECSIT in the reprogramming of bioenergetic pathways linked to Aß toxicity, a hallmark of AD.


Subject(s)
Alzheimer Disease , Electron Transport Complex I , Humans , Oxidation-Reduction , Electron Transport Complex I/metabolism , Energy Metabolism , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism
4.
Eur J Med Chem ; 260: 115771, 2023 Nov 15.
Article in English | MEDLINE | ID: mdl-37657271

ABSTRACT

Tyrosinase, a copper-containing enzyme critical in melanin biosynthesis, is a key drug target for hyperpigmentation and melanoma in humans. Testing the inhibitory effects of compounds using tyrosinase from Agaricus bisporus (AbTYR) has been a common practice to identify potential therapeutics from synthetic and natural sources. However, structural diversity among human tyrosinase (hTYR) and AbTYR presents a challenge in developing drugs that are therapeutically effective. In this study, we combined retrospective and computational analyses with experimental data to provide insights into the development of new inhibitors targeting both hTYR and AbTYR. We observed contrasting effects of Thiamidol™ and our 4-(4-hydroxyphenyl)piperazin-1-yl-derivative (6) on both enzymes; based on this finding, we aimed to investigate their binding modes in hTYR and AbTYR to identify residues that significantly improve affinity. All the information led to the discovery of compound [4-(4-hydroxyphenyl)piperazin-1-yl](2-methoxyphenyl)methanone (MehT-3, 7), which showed comparable activity on AbTYR (IC50 = 3.52 µM) and hTYR (IC50 = 5.4 µM). Based on these achievements we propose the exploitation of our computational results to provide relevant structural information for the development of newer dual-targeting molecules, which could be preliminarily tested on AbTYR as a rapid and inexpensive screening procedure before being tested on hTYR.


Subject(s)
Hyperpigmentation , Monophenol Monooxygenase , Humans , Retrospective Studies , Copper , Drug Delivery Systems , Piperazine
5.
Curr Opin Struct Biol ; 80: 102573, 2023 06.
Article in English | MEDLINE | ID: mdl-36966690

ABSTRACT

Alzheimer's disease (AD) is a progressive neurodegenerative disease with no cure where the underlying causes remain elusive. Mitochondrial dysfunction has become a prime suspect in AD pathogenesis since bioenergetic deficits precede the pathology. With advancing structural biology techniques at synchrotrons and cryo-electron microscopes, it is becoming possible to determine the structures of key proteins suspected to contribute to the initiation and propagation of AD, and investigate their interactions. In this review, we provide an overview of the recent developments concerning the structural aspects of mitochondrial protein complexes and their assembly factors involved the production of energy, in pursuit of therapies to halt or even reverse this disease in the early stages when mitochondria are most sensitive to amyloid toxicity.


Subject(s)
Alzheimer Disease , Neurodegenerative Diseases , Humans , Alzheimer Disease/metabolism , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Mitochondria/metabolism , Energy Metabolism , Amyloid/metabolism , Amyloid beta-Peptides/metabolism
6.
Nat Commun ; 13(1): 4969, 2022 08 24.
Article in English | MEDLINE | ID: mdl-36002457

ABSTRACT

To eliminate specific or aberrant transcripts, eukaryotes use nuclear RNA-targeting complexes that deliver them to the exosome for degradation. S. pombe MTREC, and its human counterpart PAXT, are key players in this mechanism but inner workings of these complexes are not understood in sufficient detail. Here, we present an NMR structure of an MTREC scaffold protein Red1 helix-turn-helix domain bound to the Iss10 N-terminus and show this interaction is required for proper cellular growth and meiotic mRNA degradation. We also report a crystal structure of a Red1-Ars2 complex explaining mutually exclusive interactions of hARS2 with various ED/EGEI/L motif-possessing RNA regulators, including hZFC3H1 of PAXT, hFLASH or hNCBP3. Finally, we show that both Red1 and hZFC3H1 homo-dimerize via their coiled-coil regions indicating that MTREC and PAXT likely function as dimers. Our results, combining structures of three Red1 interfaces with in vivo studies, provide mechanistic insights into conserved features of MTREC/PAXT architecture.


Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Carrier Proteins/metabolism , Humans , Meiosis , RNA/metabolism , RNA Stability/genetics , RNA, Messenger/metabolism , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism
7.
Sci Rep ; 11(1): 10774, 2021 05 24.
Article in English | MEDLINE | ID: mdl-34031444

ABSTRACT

Two-component systems (TCS) are widespread signaling systems present in all domains of life. TCS typically consist of a signal receptor/transducer and a response regulator. The receptors (histidine kinases, chemoreceptors and photoreceptors) are often embedded in the membrane and have a similar modular structure. Chemoreceptors were shown to function in highly ordered arrays, with trimers of dimers being the smallest functional unit. However, much less is known about photoreceptors. Here, we use small-angle scattering (SAS) to show that detergent-solubilized sensory rhodopsin II in complex with its cognate transducer forms dimers at low salt concentration, which associate into trimers of dimers at higher buffer molarities. We then fit an atomistic model of the whole complex into the SAS data. The obtained results suggest that the trimer of dimers is "tripod"-shaped and that the contacts between the dimers occur only through their cytoplasmic regions, whereas the transmembrane regions remain unconnected.

8.
Angew Chem Int Ed Engl ; 60(9): 4689-4697, 2021 02 23.
Article in English | MEDLINE | ID: mdl-33320993

ABSTRACT

Fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are mitochondrial redox processes that generate ATP. The biogenesis of the respiratory Complex I, a 1 MDa multiprotein complex that is responsible for initiating OXPHOS, is mediated by assembly factors including the mitochondrial complex I assembly (MCIA) complex. However, the organisation and the role of the MCIA complex are still unclear. Here we show that ECSIT functions as the bridging node of the MCIA core complex. Furthermore, cryo-electron microscopy together with biochemical and biophysical experiments reveal that the C-terminal domain of ECSIT directly binds to the vestigial dehydrogenase domain of the FAO enzyme ACAD9 and induces its deflavination, switching ACAD9 from its role in FAO to an MCIA factor. These findings provide the structural basis for the MCIA complex architecture and suggest a unique molecular mechanism for coordinating the regulation of the FAO and OXPHOS pathways to ensure an efficient energy production.


Subject(s)
Electron Transport Complex I/chemistry , Flavin-Adenine Dinucleotide/metabolism , Mitochondria/metabolism , Acyl-CoA Dehydrogenases/genetics , Acyl-CoA Dehydrogenases/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Cryoelectron Microscopy , Electron Transport Complex I/metabolism , Energy Metabolism , Flavin-Adenine Dinucleotide/chemistry , Humans , Oxidative Phosphorylation , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
9.
Int J Mol Sci ; 21(3)2020 Jan 30.
Article in English | MEDLINE | ID: mdl-32019241

ABSTRACT

Tyrosinase-related protein 1 (TYRP1) is one of the three human melanogenic enzymes involved in the biosynthesis of melanin, a pigment responsible for the color of the skin, hair, and eyes. It shares high sequence identity with tyrosinase, but has two zinc ions in its active site rather than two copper ions as in tyrosinase. Typical tyrosinase inhibitors do not directly coordinate to the zinc ions of TYRP1. Here, we show, from an X-ray crystal structure determination, that phenylthiourea, a highly potent tyrosinase inhibitor, does neither coordinate the active site zinc ions, but binds differently from other structurally characterized TYRP1-inhibitor complexes. Its aromatic ring is directed outwards from the active site, apparently as a result from the absence of polar oxygen substituents that can take the position of water molecules bound in the active site. The compound binds via hydrophobic interactions, thereby blocking substrate access to the active site.


Subject(s)
Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Phenylthiourea/metabolism , Catalytic Domain , Crystallography, X-Ray , Humans , Models, Molecular , Protein Conformation
10.
RSC Med Chem ; 12(3): 363-369, 2020 Nov 13.
Article in English | MEDLINE | ID: mdl-34041485

ABSTRACT

Human tyrosinase (hTYR) and tyrosinase-related protein 1 (hTYRP1) are closely-related enzymes involved in the synthesis of melanin, which are selectively expressed in melanocytes and, in a pathological context, in melanoma lesions. We used a previously described tyrosinase inhibitor (Thiamidol™) and DNA-encoded library technology for the discovery of novel hTYR and hTYRP1 ligands, that could be used as vehicles for melanoma targeting. Performing de novo selections with DNA-encoded libraries, we discovered novel ligands capable of binding to both hTYR and hTYRP1. More potent ligands were obtained by multimerizing Thiamidol™ moieties, leading to homotetrameric structures that avidly bound to melanoma cells, as revealed by flow cytometry. These findings suggest that melanoma lesions may, in the future, be targeted not only by monoclonal antibody reagents but also by small organic ligands.

11.
Forensic Sci Int ; 298: 312-315, 2019 May.
Article in English | MEDLINE | ID: mdl-30925350

ABSTRACT

V. M. E. was evacuated when he was a young boy in 1939. He left an aunt and cousins in Spain (G. E. family). He was adopted in Belgium by the D. family and thus his new name became V. D. He has been unable to remember his childhood before his adoption, a symptomatology compatible with amnesia for personal identity, presumably because he may have suffered a head contusion before or during his exodus. Identification tests were performed on blood samples from V. D. and V. G. E., a mitochondrial cousin of the missing boy. V. G. E. and the missing boy have a common mitochondrial ancestor, their maternal grandmother. The mitochondrial profile of both samples turned out to be highly specific, which allowed the genetic identification of V. D. as V. M. E. As a result, V. D. has reclaimed his past and reunited with his former family in Spain after more than seven decades. As far as we know, this is the first report describing the application of mitochondrial DNA in the identification of a person evacuated during the Spanish Civil War suffering from amnesia for personal identity.


Subject(s)
DNA Fingerprinting , DNA, Mitochondrial/genetics , Pedigree , Aged , Amnesia/complications , Armed Conflicts , Humans , Male , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Refugees , Sequence Analysis, DNA , Spain
12.
Sci Rep ; 8(1): 18008, 2018 12 20.
Article in English | MEDLINE | ID: mdl-30573755

ABSTRACT

The availability of genomic data from extinct homini such as Neanderthals has caused a revolution in palaeontology allowing the identification of modern human-specific protein substitutions. Currently, little is known as to how these substitutions alter the proteins on a molecular level. Here, we investigate adenylosuccinate lyase, a conserved enzyme involved in purine metabolism for which several substitutions in the modern human protein (hADSL) have been described to affect intelligence and behaviour. During evolution, modern humans acquired a specific substitution (Ala429Val) in ADSL distinguishing it from the ancestral variant present in Neanderthals (nADSL). We show here that despite this conservative substitution being solvent exposed and located distant from the active site, there is a difference in thermal stability, but not enzymology or ligand binding between nADSL and hADSL. Substitutions near residue 429 which do not profoundly affect enzymology were previously reported to cause neurological symptoms in humans. This study also reveals that ADSL undergoes conformational changes during catalysis which, together with the crystal structure of a hitherto undetermined product bound conformation, explains the molecular origin of disease for several modern human ADSL mutants.


Subject(s)
Adenylosuccinate Lyase/chemistry , Adenylosuccinate Lyase/genetics , Evolution, Molecular , Neanderthals/genetics , Amino Acid Sequence , Animals , Catalysis , Catalytic Domain , Crystallization , Enzyme Stability , Humans , Models, Molecular , Mutation, Missense , Protein Conformation , Social Change , Temperature
13.
Chemistry ; 24(1): 47-55, 2018 Jan 02.
Article in English | MEDLINE | ID: mdl-29052256

ABSTRACT

Melanin is the main pigment responsible for the color of human skin, hair and eye. Its biosynthesis requires three melanogenic enzymes, tyrosinase (TYR), and the tyrosinase-related proteins TYRP1 and TYRP2. The difficulty of isolating pure and homogeneous proteins from endogenous sources has hampered their study, and resulted in many contradictory findings regarding their physiological functions. In this review, we summarize recent advances on the structure and function of TYR and TYRPs by virtue of the crystal structure of human TYRP1, which is the first available structure of a mammalian melanogenic enzyme. This structure, combined with tyrosinase structures from other lower eukaryotes and mutagenesis studies of key active site residues, sheds light on the mechanism of TYR and TYRPs. Furthermore, a TYRP1-based homology model of TYR provides a high-quality platform to map and analyze albinism-related mutations, as well as the design of specific antimelanogenic compounds. Finally, we provide perspectives for future structure/function studies of TYR and TYRPs.


Subject(s)
Intramolecular Oxidoreductases/chemistry , Melanins/biosynthesis , Membrane Glycoproteins/chemistry , Monophenol Monooxygenase/chemistry , Mutagenesis/genetics , Mutation/genetics , Oxidoreductases/chemistry , Animals , Catalytic Domain , Humans , Intramolecular Oxidoreductases/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Monophenol Monooxygenase/genetics , Monophenol Monooxygenase/metabolism , Mutagenesis/physiology , Mutation/physiology , Oxidoreductases/genetics , Oxidoreductases/metabolism , Pigmentation
14.
Angew Chem Int Ed Engl ; 56(33): 9812-9815, 2017 08 07.
Article in English | MEDLINE | ID: mdl-28661582

ABSTRACT

Tyrosinase-related protein 1 (TYRP1) is one of three tyrosinase-like glycoenzymes in human melanocytes that are key to the production of melanin, the compound responsible for the pigmentation of skin, eye, and hair. Difficulties with producing these enzymes in pure form have hampered the understanding of their activity and the effect of mutations that cause albinism and pigmentation disorders. Herein we show that the typical tyrosinase-like subdomain of TYRP1 contains two zinc ions in the active site instead of copper ions as found in tyrosinases, which explains why TYRP1 does not exhibit tyrosinase redox activity. In addition, the structures reveal for the first time that the Cys-rich subdomain, which is unique to vertebrate melanogenic proteins, has an epidermal growth factor-like fold and is tightly associated with the tyrosinase subdomain. Our structures suggest that most albinism-related mutations of TYRP1 affect its stability or activity.


Subject(s)
Membrane Glycoproteins/metabolism , Oxidoreductases/metabolism , Zinc/metabolism , Binding Sites , Humans , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Models, Molecular , Mutation , Oxidoreductases/chemistry , Oxidoreductases/genetics , Protein Conformation , Zinc/chemistry
15.
Front Mol Biosci ; 3: 43, 2016.
Article in English | MEDLINE | ID: mdl-27597947

ABSTRACT

Neurons are extremely energy demanding cells and highly dependent on the mitochondrial oxidative phosphorylation (OXPHOS) system. Mitochondria generate the energetic potential via the respiratory complexes I to IV, which constitute the electron transport chain (ETC), together with complex V. These redox reactions release energy in the form of ATP and also generate reactive oxygen species (ROS) that are involved in cell signaling but can eventually lead to oxidative stress. Complex I (CI or NADH:ubiquinone oxidoreductase) is the largest ETC enzyme, containing 44 subunits and the main contributor to ROS production. In recent years, the structure of the CI has become available and has provided new insights into CI assembly. A number of chaperones have been identified in the assembly and stability of the mature holo-CI, although they are not part of its final structure. Interestingly, CI dysfunction is the most common OXPHOS disorder in humans and defects in the CI assembly process are often observed. However, the dynamics of the events leading to CI biogenesis remain elusive, which precludes our understanding of how ETC malfunctioning affects neuronal integrity. Here, we review the current knowledge of the structural features of CI and its assembly factors and the potential role of CI misassembly in human disorders such as Complex I Deficiencies or Alzheimer's and Parkinson's diseases.

16.
PLoS One ; 11(8): e0161697, 2016.
Article in English | MEDLINE | ID: mdl-27551823

ABSTRACT

Human tyrosinase (TYR) is a glycoprotein that initiates the first two reactions in the melanin biosynthesis pathway. Mutations in its encoding gene cause Oculocutaneous Albinism type I (OCA1), the most severe form of albinism, which is a group of autosomal recessive disorders characterized by reduced or absent production of melanin in skin, hair and eyes. Despite extensive structural and characterization studies of its homologues in lower eukaryotic organisms, the catalytic mechanism of human TYR and the molecular basis of OCA1 are largely unknown. In this work, we have carried out a large-scale recombinant expression of TYR that has enabled us to obtain high yields of pure and active protein, required for crystallization trials and screening of skin whitening agents, which is highly demanded in the cosmetic industry. Addition of an N-terminal honeybee melittin signal peptide for secretion of the produced protein into the (protein-free) medium, as well as a cleavable His-tag at the C-terminus, was crucial for increasing the yield of pure protein. We have successfully crystallized two TYR variants, in both glycosylated and deglycosylated forms, showing preliminary X-ray diffraction patterns at 3.5 Å resolution. Hence, we have established an expression and purification protocol suitable for the crystal structure determination of human TYR, which will give unique atomic insight into the nature and conformation of the residues that shape the substrate binding pocket that will ultimately lead to efficient compound design.


Subject(s)
Monophenol Monooxygenase/chemistry , Monophenol Monooxygenase/isolation & purification , Recombinant Proteins , Amino Acid Sequence , Animals , Chromatography , Gene Expression , Glycosylation , Humans , Monophenol Monooxygenase/genetics , Monophenol Monooxygenase/metabolism , Protein Stability , Structure-Activity Relationship , Temperature , X-Ray Diffraction
17.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 3): 244-50, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26919530

ABSTRACT

Mushroom tyrosinase-associated lectin-like protein (MtaL) binds to mature Agaricus bisporus tyrosinase in vivo, but the exact physiological function of MtaL is unknown. In this study, the crystal structure of recombinant MtaL is reported at 1.35 Å resolution. Comparison of its structure with that of the truncated and cleaved MtaL present in the complex with tyrosinase directly isolated from mushroom shows that the general ß-trefoil fold is conserved. However, differences are detected in the loop regions, particularly in the ß2-ß3 loop, which is intact and not cleaved in the recombinant MtaL. Furthermore, the N-terminal tail is rotated inwards, covering the tyrosinase-binding interface. Thus, MtaL must undergo conformational changes in order to bind mature mushroom tyrosinase. Very interestingly, the ß-trefoil fold has been identified to be essential for carbohydrate interaction in other lectin-like proteins. Comparison of the structures of MtaL and a ricin-B-like lectin with a bound disaccharide shows that MtaL may have a similar carbohydrate-binding site that might be involved in glycoreceptor activity.


Subject(s)
Agaricus , Fungal Proteins/chemistry , Lectins/chemistry , Amino Acid Sequence , Binding Sites , Crystallization , Crystallography, X-Ray , Monophenol Monooxygenase/chemistry , Protein Binding , Protein Domains , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid
18.
Sci Rep ; 6: 19729, 2016 Jan 28.
Article in English | MEDLINE | ID: mdl-26818620

ABSTRACT

Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity.


Subject(s)
Cell Cycle/physiology , DNA Replication/physiology , DNA, Fungal/biosynthesis , Nucleosomes/metabolism , Saccharomyces cerevisiae/metabolism , DNA, Fungal/genetics , Nucleosomes/genetics , Saccharomyces cerevisiae/genetics
19.
J Mol Biol ; 427(6 Pt B): 1436-1450, 2015 Mar 27.
Article in English | MEDLINE | ID: mdl-25640309

ABSTRACT

Despite the remarkable progress achieved in the identification of specific genes involved in breast cancer (BC), our understanding of their complex functioning is still limited. In this manuscript, we systematically explore the existence of direct physical interactions between the products of BC core and associated genes. Our aim is to generate a protein interaction network of BC-associated gene products and suggest potential molecular mechanisms to unveil their role in the disease. In total, we report 599 novel high-confidence interactions among 44 BC core, 54 BC candidate/associated and 96 newly identified proteins. Our findings indicate that this network-based approach is indeed a robust inference tool to pinpoint new potential players and gain insight into the underlying mechanisms of those proteins with previously unknown roles in BC. To illustrate the power of our approach, we provide initial validation of two BC-associated proteins on the alteration of DNA damage response as a result of specific re-wiring interactions. Overall, our BC-related network may serve as a framework to integrate clinical and molecular data and foster novel global therapeutic strategies.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Computational Biology/methods , Gene Regulatory Networks , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Protein Interaction Maps , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cells, Cultured , DNA Damage/genetics , Female , Fluorescent Antibody Technique , Genetic Predisposition to Disease , Humans , Immunoprecipitation , Oligonucleotide Array Sequence Analysis , Two-Hybrid System Techniques
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