Dynamic Processing of a Common Oxidative DNA Lesion by the First Two Enzymes of the Base Excision Repair Pathway

Base excision repair (BER) is the primary pathway by which eukaryotic cells resolve single base damage. One common example of single base damage is 8-oxo-7,8-dihydro-2ʹ-deoxoguanine (8-oxoG). High incidence and mutagenic potential of 8-oxoG necessitate rapid and efficient DNA repair. How BER enzymes coordinate their activities to resolve 8-oxoG damage while limiting cytotoxic BER intermediates from propagating genomic instability remains unclear. Here we use single-molecule Förster resonance energy transfer (smFRET) and ensemble-level techniques to characterize the activities and interactions of consecutive BER enzymes important for repair of 8-oxoG. In addition to characterizing the damage searching and processing mechanisms of human 8-oxoguanine glycosylase 1 (hOGG1), our data support the existence of a ternary complex between hOGG1, the damaged DNA substrate, and human AP endonuclease 1 (APE1). Our results indicate that hOGG1 is actively displaced from its abasic site containing product by protein–protein interactions with APE1 to ensure timely repair of damaged DNA.     

Targeting the DNA damage response (DDR) by natural compounds

Natural compounds (NC) are an important source of anticancer drugs. The genomic DNA of tumor cells is a major target of conventional anticancer therapeutics (cAT). DNA damage elicits a complex stress response programme termed DNA damage response (DDR), with the PI3-like kinase ATM and ATR being the key regulators. Since the DDR coordinates mechanisms of DNA repair and apoptosis, hence regulating the balance between death and survival, it is an attractive target of novel anticancer strategies. The aim of the study was to identify natural compounds derived from endophytic fungi, lichens, marine sponges or plants that interfere with mechanisms of the DDR. To this end, the cytotoxic and DDR modulating potency of 296 natural compounds, used alone or in combination with the cAT cisplatin (Cis) and doxorubicin (Doxo) was investigated by fluorescence-based analysis of the ATM/ATR-catalyzed S139 phosphorylation of histone 2AX (γH2AX), a surrogate marker of DNA damage-triggered DDR. After initial screening, a total of ten natural compounds were identified that were toxic in pancreatic carcinoma cells and activated the DDR on their own and/or promoted the DDR if used in combination with cAT. Their mode of action was shown to be independent of drug transport mechanisms. Based on their chemical structures, DDR modulatory activity and published data we suggest the marine NC 5-epi-nakijiquinone Q and 5-epi-ilimaquinone as well as the fungal compound secalonic acid F as most promising NC-based drug candidates for future synthesis of DDR-modulating chemical derivatives and their preclinical in vitro and in vivo testing.     

Active and Passive Destabilization of G-Quadruplex DNA by the Telomere POT1-TPP1 Complex

Chromosome ends are protected by guanosine-rich telomere DNA that forms stable G-quadruplex (G4) structures. The heterodimeric POT1-TPP1 complex interacts specifically with telomere DNA to shield it from illicit DNA damage repair and to resolve secondary structure that impedes telomere extension. The mechanism by which POT1-TPP1 accomplishes these tasks is poorly understood. Here, we establish the kinetic framework for POT1-TPP1 binding and unfolding of telomere G4 DNA. Our data identify two modes of POT1-TPP1 destabilization of G4 DNA that are governed by protein concentration. At low concentrations, POT1-TPP1 passively captures transiently unfolded G4s. At higher concentrations, POT1-TPP1 proteins bind to G4s to actively destabilize the DNA structures. Cancer-associated POT1-TPP1 mutations impair multiple reaction steps in this process, resulting in less efficient destabilization of G4 structures. The mechanistic insight highlights the importance of cell cycle dependent expression and localization of the POT1-TPP1 complex and distinguishes diverse functions of this complex in telomere maintenance.     

Deciphering Spatial Protein–Protein Interactions in Brain Using Proximity Labeling

Cellular biomolecular complexes including protein–protein, protein–RNA, and protein–DNA interactions regulate and execute most biological functions. In particular in brain, protein–protein interactions (PPIs) mediate or regulate virtually all nerve cell functions, such as neurotransmission, cell–cell communication, neurogenesis, synaptogenesis, and synaptic plasticity. Perturbations of PPIs in specific subsets of neurons and glia are thought to underly a majority of neurobiological disorders. Therefore, understanding biological functions at a cellular level requires a reasonably complete catalog of all physical interactions between proteins. An enzyme-catalyzed method to biotinylate proximal interacting proteins within 10 to 300 nm of each other is being increasingly used to characterize the spatiotemporal features of complex PPIs in brain. Thus, proximity labeling has emerged recently as a powerful tool to identify proteomes in distinct cell types in brain as well as proteomes and PPIs in structures difficult to isolate, such as the synaptic cleft, axonal projections, or astrocyte–neuron junctions. In this review, we summarize recent advances in proximity labeling methods and their application to neurobiology.     

Recent insight into intermediate filament structure

Intermediate filaments (IFs) are key players in multiple cellular processes throughout human tissues. Their biochemical and structural properties are important for understanding filament assembly mechanisms, for interactions between IFs and binding partners, and for developing pharmacological agents that target IFs. IF proteins share a conserved coiled-coil central-rod domain flanked by variable N-terminal ‘head’ and C-terminal ‘tail’ domains. There have been several recent advances in our understanding of IF structure from the study of keratins, glial fibrillary acidic protein, and lamin. These include discoveries of (i) a knob–pocket tetramer assembly mechanism in coil 1B; (ii) a lamin-specific coil 1B insert providing a one-half superhelix turn; (iii) helical, yet flexible, linkers within the rod domain; and (iv) the identification of coil 2B residues required for mature filament assembly. Furthermore, the head and tail domains of some IFs contain low-complexity aromatic-rich kinked segments, and structures of IFs with binding partners show electrostatic surfaces are a major contributor to complex formation. These new data advance the connection between IF structure, pathologic mutations, and clinical diseases in humans.     

Past,present, and perspectives of protein N-terminal methylation

The posttranslational methylation of the α-N-terminal amino group of proteins was first documented over 40 years ago, but the functional significance of this modification has been underexplored relative to lysine and arginine methylation. Increasing reports implicates α-N-terminal methylation as a widespread and critical regulator of mitosis, chromatin interactions, DNA repair, and translation fidelity. Here, we summarize advances in the current understanding of protein α-N-terminal methylation biological functions and mechanisms across eukaryotic organisms. Also, we describe the recent literature on substrate recognition and the discovery of potent and selective inhibitors for protein N-terminal methyltransferases. Finally, we summarize the emergent crosstalk between α-N-terminal methylation and other N-terminal modifications.     

A novel KU70-mutant human leukemic cell line generated using CRISPR-Cas9 shows increased sensitivity to DSB inducing agents and reduced NHEJ activity

KU70 (XRCC6 gene in humans) is one of the proteins in the KU70-KU80 heterodimer which is the first component recruited to broken DNA ends during DNA double-strand break repair through nonhomologous end joining (NHEJ). Previous studies have shown that Ku70 deficient mouse cells are defective in NHEJ and V(D)J recombination. In contrast, heterozygous KU70 mutant human cell lines did not show any significant change in cell viability and sensitivity towards ionizing radiation. In this study, we used CRISPR-Cas9 technique to generate a KU70 mutant (heterozygous) human pre-B leukemic cell line (N6-KU70–2-DG). We observed that the N6-KU70–2-DG cells showed a prominent reduction in the expression of both KU70 mRNA and protein. The mutant cells showed reduced cell viability, increased sensitivity to DSB inducing agents such as ionizing radiation (IR) and etoposide, and increased number of unrepaired DSBs after exposure to IR. In addition, the mutant cells showed a reduction in the NHEJ activity and increased rate of microhomology mediated joining (MMEJ) activity. KU70 mutant cells also revealed enhanced level of senescence markers following irradiation. Thus, we report a novel KU70-mutant leukemic cell line (heterozygous) with reduced NHEJ, which is sensitive to DNA damaging agents, unlike the previously reported other KU heterozygous mutant cell lines.     

Inhibition of NADPH oxidase alleviates germ cell apoptosis and ER stress during testicular ischemia reperfusion injury

Testicular torsion and detorsion (TTD) is a serious urological condition affecting young males that is underlined by an ischemia reperfusion injury (tIRI) to the testis as the pathophysiological mechanism. During tIRI, uncontrolled production of oxygen reactive species (ROS) causes DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to explore whether inhibition of NADPH oxidase (NOX), a major source of intracellular ROS, will prevent tIRI-induced GCA and its association with endoplasmic reticulum (ER) stress. Sprague-Dawley rats (n = 36) were divided into three groups: sham, tIRI only and tIRI treated with apocynin (a NOX inhibitor). Rats undergoing tIRI endured an ischemic injury for 1 h followed by 4 h of reperfusion. Spermatogenic damage was evaluated histologically, while cellular damages were assessed using real time PCR, immunofluorescence staining, Western blot and biochemical assays. Disrupted spermatogenesis was associated with increased lipid and protein peroxidation and decreased antioxidant activity of the enzyme superoxide dismutase (SOD) as a result of tIRI. In addition, increased DNA double strand breaks and formation of 8-OHdG adducts associated with increased phosphorylation of the DNA damage response (DDR) protein H2AX. The ASK1/JNK apoptosis signaling pathway was also activated in response to tIRI. Finally, increased immuno-expression of the unfolded protein response (UPR) downstream targets: GRP78, eIF2-α1, CHOP and caspase 12 supported the presence of ER stress. Inhibition of NOX by apocynin protected against tIRI-induced GCA and ER stress. In conclusion, NOX inhibition minimized tIRI-induced intracellular oxidative damages leading to GCA and ER stress.     

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.     

ProMetheusDB: An In-Depth Analysis of the High-Quality Human Methyl-proteome

Protein arginine (R) methylation is a post-translational modification involved in various biological processes, such as RNA splicing, DNA repair, immune response, signal transduction, and tumor development. Although several advancements were made in the study of this modification by mass spectrometry, researchers still face the problem of a high false discovery rate. We present a dataset of high-quality methylations obtained from several different heavy methyl stable isotope labeling with amino acids in cell culture experiments analyzed with a machine learning–based tool and show that this model allows for improved high-confidence identification of real methyl-peptides. Overall, our results are consistent with the notion that protein R methylation modulates protein–RNA interactions and suggest a role in rewiring protein–protein interactions, for which we provide experimental evidence for a representative case (i.e., NONO [non-POU domain–containing octamer-binding protein]–paraspeckle component 1 [PSPC1]). Upon intersecting our R-methyl-sites dataset with the PhosphoSitePlus phosphorylation dataset, we observed that R methylation correlates differently with S/T-Y phosphorylation in response to various stimuli. Finally, we explored the application of heavy methyl stable isotope labeling with amino acids in cell culture to identify unconventional methylated residues and successfully identified novel histone methylation marks on serine 28 and threonine 32 of H3. The database generated, named ProMetheusDB, is freely accessible at https://bioserver.ieo.it/shiny/app/prometheusdb.     

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.     

RPA-like single-stranded DNA-binding protein complexes including CST serve as specialized processivity factors for polymerases

Telomeres and other single-stranded regions of the genome require specialized management to maintain stability and for proper progression of DNA metabolism pathways. Human Replication Protein A and CTC1-STN1-TEN1 are structurally similar heterotrimeric protein complexes that have essential ssDNA-binding roles in DNA replication, repair, and telomeres. Yeast and ciliates have related ssDNA-binding proteins with strikingly conserved structural features to these human heterotrimeric protein complexes. Recent breakthrough structures have extended our understanding of these commonalities by illuminating a common mechanism used by these proteins to act as processivity factors for their partner polymerases through their ability to manage ssDNA.