Absolute quantification of DNA damage response proteins.

Background: DNA damage response (DDR) and repair are vital for safeguarding genetic information and ensuring the survival and accurate transmission of genetic material. DNA damage, such as DNA double-strand breaks (DSBs), triggers a response where sensor proteins recognize DSBs. Information is transmitted to kinases, initiating a sequence resulting in the activation of the DNA damage response and recruitment of other DDR and repair proteins to the DSB site in a highly orderly sequence.

Machine learning extracts oncogenic-specific γ-H2AX foci formation pattern upon genotoxic stress.

H2AX is a histone H2A variant that becomes phosphorylated upon genotoxic stress. The phosphorylated H2AX (γ-H2AX) plays an antioncogenic role in the DNA damage response and its foci patterns are highly variable, in terms of intensities and sizes. However, whether characteristic γ-H2AX foci patterns are associated with oncogenesis (oncogenic-specific γ-H2AX foci patterns) remains unknown.

Impact of Nuclear NAD Synthesis via Histone Dynamics on DNA Repair during Cellular Senescence To Prevent Tumorigenesis.

NAD synthesis is a fundamental process in living cells. The effects of local metabolite production on chromatin influence the epigenetic status of chromatin in DNA metabolism. We have previously shown that K5 acetylation of H2AX by TIP60 is required for the ADP ribosylation activity of PARP-1, for histone H2AX exchange at DNA damage sites.

Creation of Cross-Linked Crystals With Intermolecular Disulfide Bonds Connecting Symmetry-Related Molecules Allows Retention of Tertiary Structure in Different Solvent Conditions.

Protein crystals are generally fragile and sensitive to subtle changes such as pH, ionic strength, and/or temperature in their crystallization mother liquor. Here, using T4 phage lysozyme as a model protein, the three-dimensional rigidification of protein crystals was conducted by introducing disulfide cross-links between neighboring molecules in the crystal. The effect of cross-linking on the stability of the crystals was evaluated by microscopic observation and X-ray diffraction.

Structural Analysis of Hen Egg Lysozyme Refolded after Denaturation at Acidic pH.

Protein structures fluctuate in solution; therefore, proteins have multiple stable structures that are slightly different from each other. In this study, we determined the crystal structure of hen egg lysozyme refolded after denaturation at acidic pH (rHEL) and found a structure different from native HEL (nHEL). The different local conformations of the peptide bond between Asp101 and Gly102 found in the crystal structure was supported by the NMR results for nHEL and rHEL.

HMGB1 signaling phosphorylates Ku70 and impairs DNA damage repair in Alzheimer's disease pathology.

DNA damage is increased in Alzheimer's disease (AD), while the underlying mechanisms are unknown. Here, we employ comprehensive phosphoproteome analysis, and identify abnormal phosphorylation of 70 kDa subunit of Ku antigen (Ku70) at Ser77/78, which prevents Ku70-DNA interaction, in human AD postmortem brains. The abnormal phosphorylation inhibits accumulation of Ku70 to the foci of DNA double strand break (DSB), impairs DNA damage repair and eventually causes transcriptional repression-induced atypical cell death (TRIAD).

Hepta-Histidine Inhibits Tau Aggregation.

Tau aggregation is a central hallmark of tauopathies such as frontotemporal lobar degeneration and progressive supranuclear palsy as well as of Alzheimer's disease, and it has been a target for therapeutic development. Herein, we unexpectedly found that hepta-histidine (7H), an inhibitor of the interaction between Ku70 and Huntingtin proteins, suppresses aggregation of Tau-R3 peptides . Addition of the trans-activator of transcription (TAT) sequence (YGRKKRRQRRR) derived from the TAT protein to 7H increased its permeability into cells, and TAT-7H treatment of iPS cell-derived neurons carrying Tau or APP mutations suppressed Tau phosphorylation.

Protocol for BCR-mediated plasma cell differentiation and purification of chromatin-associated proteins.

Molecular-level understanding of plasma cell (PC) differentiation has been modeled using lipopolysaccharide (LPS) stimulation . However, this system does not involve the B-cell receptor (BCR)-a critical component of B cell biology. Here, we present a protocol for PC differentiation system dependent on BCR signaling that easily scales up for cell number-demanding applications, including protein complex purification.

Functional impacts of the ubiquitin-proteasome system on DNA damage recognition in global genome nucleotide excision repair.

The ubiquitin-proteasome system (UPS) plays crucial roles in regulation of various biological processes, including DNA repair. In mammalian global genome nucleotide excision repair (GG-NER), activation of the DDB2-associated ubiquitin ligase upon UV-induced DNA damage is necessary for efficient recognition of lesions. To date, however, the precise roles of UPS in GG-NER remain incompletely understood.

Mutational effects of Cys113 on structural dynamics of Pin1.

Pin1 is a peptidyl-prolyl isomerase (PPIase) which catalyzes isomerization of pS/pT-P bond. Its activity is related to various cellular functions including suppression of Alzheimer's disease. A cysteine residue C113 is known to be important for its PPIase activity; a mutation C113A reduced the activity by 130-fold.

Development of novel lithocholic acid derivatives as vitamin D receptor agonists.

Lithocholic acid (2) was identified as the second endogenous ligand of vitamin D receptor (VDR), though its binding affinity to VDR and its vitamin D activity are very weak compared to those of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1). 3-Acylated lithocholic acids were reported to be slightly more potent than lithocholic acid (2) as VDR agonists. Here, aiming to develop more potent lithocholic acid derivatives, we synthesized several derivatives bearing a 3-sulfonate/carbonate or 3-amino/amide substituent, and examined their differentiation-inducing activity toward human promyelocytic leukemia HL-60 cells.

Epigenetic interplays between DNA demethylation and histone methylation for protecting oncogenesis.

Epigenetic systems are organized by different types of modifications on histones and DNA. To determine how epigenetic systems can produce variable, yet stable cellular outcomes, understanding the collaboration between these modifications is the key. A recent study by Yamagata and Kobayashi revealed the direct interplay between the regulation of two epigenetic modifications: DNA de-methylation by TET2 and histone H3-K36 methylation.

Dual conformation of the ligand induces the partial agonistic activity of retinoid X receptor α (RXRα).

1-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino]benzotriazole-5-carboxylic acid (CBt-PMN), a partial agonist of retinoid X receptor (RXR), has attracted attention due to its potential to treat type 2 diabetes and central nervous system diseases with reduced adverse effects of existing full agonists. Herein, we report the crystal structure of CBt-PMN-bound ligand-binding domain of human RXRα (hRXRα) and its biochemical characterization. Interestingly, the structure is a tetramer in nature, in which CBt-PMNs are clearly found binding in two different conformations.

The trans isomer of Tau peptide is prone to aggregate, and the WW domain of Pin1 drastically decreases its aggregation.

In Alzheimer's, the disease-related protein Tau is hyperphosphorylated and aggregates into neurofibrillary tangles (NFT). The cis isomer of the phosphorylated Thr231-Pro232 has been proposed as a precursor of aggregation ('Cistauosis'), but this aggregation scheme is not yet completely accepted. Here, we synthesized peptides comprising a phosphorylated region including Thr231-Pro232 and an aggregation-core region R1 to investigate isomer-specific-aggregation of Tau.

Cancer-associated mutations of histones H2B, H3.1 and H2A.Z.1 affect the structure and stability of the nucleosome.

Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro.

25 S-Adamantyl-23-yne-26,27-dinor-1α,25-dihydroxyvitamin D: Synthesis, Tissue Selective Biological Activities, and X-ray Crystal Structural Analysis of Its Vitamin D Receptor Complex.

Both 25 R- and 25 S-25-adamantyl-23-yne-26,27-dinor-1α,25-dihydroxyvitamin D (4a and 4b) were stereoselectively synthesized by a Pd(0)-catalyzed ring closure and Suzuki-Miyaura coupling between enol-triflate 7 and alkenyl-boronic ester 8. The 25 S isomer (4b) showed high vitamin D receptor (VDR) affinity (50% of that of the natural hormone 1α,25-dihydroxyvitamin D, 1) and transactivation potency (kidney HEK293, 90%). In endogenous gene expression, it showed high cell-type selectivity for kidney cells (HEK293, CYP24A1 160% of 1), bone cells (MG63, osteocalcin 64%), and monocytes (U937, CAMP 96%) over intestine (SW480, CYP24A1 8%) and skin (HaCaT, CYP24A1 7%) cells.

Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations.

Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities.

An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior.

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet.

The CDK-PLK1 axis targets the DNA damage checkpoint sensor protein RAD9 to promote cell proliferation and tolerance to genotoxic stress.

Genotoxic stress causes proliferating cells to activate the DNA damage checkpoint, to assist DNA damage recovery by slowing cell cycle progression. Thus, to drive proliferation, cells must tolerate DNA damage and suppress the checkpoint response. However, the mechanism underlying this negative regulation of checkpoint activation is still elusive.

SUMO modification system facilitates the exchange of histone variant H2A.Z-2 at DNA damage sites.

Histone exchange and histone post-translational modifications play important roles in the regulation of DNA metabolism, by re-organizing the chromatin configuration. We previously demonstrated that the histone variant H2A.Z-2 is rapidly exchanged at damaged sites after DNA double strand break induction in human cells.

Regional cerebral glucose metabolism in systemic lupus erythematosus patients with major depressive disorder.

Objectives: Depression is frequently observed in patients with systemic lupus erythematosus (SLE). Neuropsychiatric SLE (NPSLE) patients often exhibit cerebral hypometabolism, but the association between cerebral metabolism and depression remains unclear. To elucidate the features of cerebral metabolism in SLE patients with depression, we performed brain 18F-fluoro-d-glucose positron emission tomography (FDG-PET) on SLE patients with and without major depressive disorder.

Halophilic mechanism of the enzymatic function of a moderately halophilic dihydrofolate reductase from Haloarcula japonica strain TR-1.

Dihydrofolate (DHF) reductase coded by a plasmid of the extremely halophilic archaeon Haloarcula japonica strain TR-1 (HjDHFR P1) shows moderate halophilicity on enzymatic activity at pH 6.0, although there is no significant effect of NaCl on its secondary structure. To elucidate the salt-activation and -inactivation mechanisms of this enzyme, we investigated the effects of pH and salt concentration, deuterium isotope effect, steady-state kinetics, and rapid-phase ligand-binding kinetics.