Tandem Affinity Purification and Mass-Spectrometric Analysis of FACT and Associated Proteins.

Isolation of a protein/complex is important for its biochemical and structural characterization with mechanistic insights. TAP (tandem affinity purification) strategy allows rapid isolation of cellular proteins/complexes with a high level of purity. This methodology involves an immuno-affinity-based purification followed by a conformation-based isolation to obtain a highly homogeneous protein/complex. Here, we describe the TAP-mediated isolation of endogenous FACT (facilitates chromatin transcription; a heterodimer), an essential histone chaperone associated with BER (base excision repair). However, it is not clearly understood how FACT regulates BER. Such knowledge would advance our understanding of BER with implications in disease pathogenesis, since BER is an evolutionarily conserved process that is linked to various diseases including ageing, neurodegenerative disorders, and cancers. Using isolated FACT by TAP methodology, one can study the mechanisms of action of FACT in BER. Further, isolated FACT can be used for studies in other DNA transactions such as transcription and replication, as FACT is involved in these processes. Furthermore, TAP-mediated isolation strategy can be combined with mass spectrometry to identify the protein interaction partners of FACT.

Fast and efficient DNA replication with purified human proteins.

Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS-TIPIN (respectively known as Ctf4, Mrc1 and Tof1-Csm3 in Saccharomyces cerevisiae) are organized around the CDC45-MCM-GINS (CMG) replicative helicase1-7. Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18-RFC. We show how CLASPIN and TIMELESS-TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome8, AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα9,10, the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes.

Identification of Optimal Expression Parameters and Purification of a Codon-Optimized Human GLIS1 Transcription Factor from Escherichia coli.

GLIS1 has multiple roles in embryonic development and in deriving induced pluripotent stem cells by aiding signaling pathways and chromatin assembly. An inexpensive and simple method to produce human GLIS1 protein from Escherichia coli (E. coli) is demonstrated in this study. Various parameters such as codon usage bias, E. coli strains, media, induction conditions (such as inducer concentration, cell density, time, and temperature), and genetic constructs were investigated to obtain soluble expression of human GLIS1 protein. Using identified expression conditions and an appropriate genetic construct, the human GLIS1 protein was homogeneously purified (purity > 90%) under native conditions. Importantly, the purified protein has upheld a stable secondary structure, as demonstrated by circular dichroism spectroscopy. To the best of our knowledge, this is the first study to report the ideal expression conditions of human GLIS1 protein in E. coli to achieve soluble expression and purification under native conditions, upholding its stable secondary structure post-purification. The biological activity of the purified GLIS1 fusion protein was further assessed in MDA-MB-231 cells. This biologically active human GLIS1 protein potentiates new avenues to understand its molecular mechanisms in different cellular functions in various cancers and in the generation of induced pluripotent stem cells.

Control of Chromatin Organization and Chromosome Behavior during the Cell Cycle through Phase Separation.

Phase-separated condensates participate in various biological activities. Liquid-liquid phase separation (LLPS) can be driven by collective interactions between multivalent and intrinsically disordered proteins. The manner in which chromatin-with various morphologies and activities-is organized in a complex and small nucleus still remains to be fully determined. Recent findings support the claim that phase separation is involved in the regulation of chromatin organization and chromosome behavior. Moreover, phase separation also influences key events during mitosis and meiosis. This review elaborately dissects how phase separation regulates chromatin and chromosome organization and controls mitotic and meiotic chromosome behavior.

Isolation of Nucleoid Fraction from Mycoplasma gallisepticum Cells with Synchronized Cell Division.

It is assumed that unknown mechanisms can be involved in adaptation Mycoplasma gallisepticum to unfavorable factors, one of these can be local rearrangements of the structure and spatial organization of the chromosome. To study these mechanisms, we obtained a culture of M. gallisepticum with synchronized division and isolated the nucleoid fraction from this culture by the method of mild cell lysis and centrifugation in a sucrose gradient. Liquid chromatography-mass spectrometry analysis of the proteome showed that in comparison with the cell lysate, the nucleoid fraction was enriched with DNA-binding proteins. This analysis will help to find new nucleoid-associated proteins and to study their dynamics, distribution, and their role during infection and under stress conditions.

Bile Salts Promote ToxR Regulon Activation during Growth under Virulence-Inducing Conditions.

Cholera is an epidemic disease caused by the Gram-negative bacterium Vibrio cholerae. V. cholerae is found in aquatic ecosystems and infects people through the consumption of V. cholerae-contaminated food or water. Following ingestion, V. cholerae responds to host cues to activate the expression of critical virulence genes that are under the control of a hierarchical regulatory system called the ToxR regulon. The ToxR regulon is tightly regulated and is expressed in vitro only under special growth conditions referred to as AKI conditions. AKI conditions have been instrumental in elucidating V. cholerae virulence regulation, but the chemical cues within AKI medium that activate virulence gene expression are unknown. In this study, we fractionated AKI medium on a reverse-phase chromatography column (RPCC) and showed that the virulence-activating molecules were retained on the RPCC column and recovered in the eluate. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of the eluate revealed the presence of a known ToxR regulon activator, taurocholate, and other bile salts. The RPCC eluate activated the ToxR regulon when added to noninducing medium and promoted TcpP dimerization in a two-hybrid system, consistent with taurocholate being responsible for the virulence-inducing activity of AKI medium. Additional experiments using purified bile salts showed that the ToxR regulon was preferentially activated in response to primary bile acids. The results of this study shed light on the chemical cues involved in V. cholerae virulence activation and suggested that V. cholerae virulence genes are modulated in response to regionally specific bile acid species in the intestine.

Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast.

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.

Cytochrome C with peroxidase-like activity encapsulated inside the small DPS protein nanocage.

Nature utilizes self-assembled protein-based structures as subcellular compartments in prokaryotes to sequester catalysts for specialized biochemical reactions. These protein cage structures provide unique isolated environments for the encapsulated enzymes. Understanding these systems is useful in the bioinspired design of synthetic catalytic organelle-like nanomaterials. The DNA binding protein from starved cells (Dps), isolated from Sulfolobus solfataricus, is a 9 nm dodecameric protein cage making it the smallest known naturally occurring protein cage. It is naturally over-expressed in response to oxidative stress. The small size, natural biodistribution to the kidney, and ability to cross the glomerular filtration barrier in in vivo experiments highlight its potential as a synthetic antioxidant. Cytochrome C (CytC) is a small heme protein with peroxidase-like activity involved in the electron transport chain and also plays a critical role in cellular apoptosis. Here we report the encapsulation of CytC inside the 5 nm interior cavity of Dps and demonstrate the catalytic activity of the resultant Dps nanocage with enhanced antioxidant behavior. The small cavity can accommodate a single CytC and this was achieved through self-assembly of chimeric cages comprising Dps subunits and a Dps subunit to which the CytC was fused. For selective isolation of CytC containing Dps cages, we utilized engineered polyhistidine tag present only on the enzyme fused Dps subunits (6His-Dps-CytC). The catalytic activity of encapsulated CytC was studied using guaiacol and 3,3',5,5'-tetramethylbenzidine (TMB) as two different peroxidase substrates and compared to the free (unencapsulated) CytC activity. The encapsulated CytC showed better pH dependent catalytic activity compared to free enzyme and provides a proof-of-concept model to engineer these small protein cages for their potential as catalytic nanoreactors.

Expression, Purification, and Solution-State NMR Analysis of the Two Human Single-Stranded DNA-Binding Proteins hSSB1 (NABP2/OBFC2B) and hSSB2 (NAPB1/OBFC2A).

Single-stranded DNA-binding proteins (SSBs) are essential to all living organisms as protectors and guardians of the genome. Apart from the well-characterized RPA, humans have also evolved two further SSBs, termed hSSB1 and hSSB2. Over the last few years, we have used NMR spectroscopy to determine the molecular and structural details of both hSSBs and their interactions with DNA and RNA. Here we provide a detailed overview of how to express and purify recombinant versions of these important human proteins for the purpose of detailed structural analysis by high-resolution solution-state NMR.

Multiple assay systems to analyze the dynamics of mitochondrial nucleoids in living mammalian cells.

BACKGROUND: Mitochondria, which play a critical role in energy production by oxidative respiration, are highly dynamic organelles and their double membranes undergo frequent events of fusion and fission. Mitochondria are believed to be derived from the endosymbiosis of proteobacteria, and thus mitochondria still contain their own DNA, mitochondrial DNA (mtDNA). Several copies of mtDNA form mitochondrial nucleoid with DNA-binding proteins. Recently, the morphology and distribution of the mitochondrial membrane and nucleoid were reported to be cooperatively regulated during their dynamic movement. However, the molecular mechanism is unclear, because the involved molecules are poorly understood, and suitable techniques to analyze nucleoid have not been fully developed. RESULTS: To solve these issues, we examined the molecular mechanism of nucleoid dynamics by two approaches. First, we constructed a new probe to perform live imaging of nucleoid dynamics using the DNA-binding domain of mitochondrial transcriptional factor A (TFAM) and the photo-convertible fluorescent protein Kikume Green-Red (KikGR). Nucleoids were visualized stably for a long period using the new probe. Second, we searched for nucleoid regulatory factors by small interfering RNA screening using HeLa cells and identified a subset of MARCH family ubiquitin ligases that affect nucleoid morphology. CONCLUSION: The factors and probe, reported in this study, would be useful to reveal novel mechanisms of mitochondrial regulation. GENERAL SIGNIFICANCE: The mtDNA dynamics should be concerned in the regulation of mitochondrial activity and its quality control, associated with mitochondrial membrane dynamics.

Single-molecule multiplexed profiling of protein-DNA complexes using magnetic tweezers.

Epigenetics, such as the dynamic interplay between DNA methylation and demethylation, play diverse roles in critical cellular events. Enzymatic activity at CpG sites, where cytosines are methylated or demethylated, is known to be influenced by the density of CpGs, methylation states, and the flanking sequences of a CpG site. However, how the relevant enzymes are recruited to and recognize their target DNA is less clear. Moreover, although DNA-binding epigenetic enzymes are ideal targets for therapeutic intervention, these targets have been rarely exploited. Single-molecule techniques offer excellent capabilities to probe site-specific protein-DNA interactions and unravel the dynamics. Here, we develop a single-molecule approach that allows multiplexed profiling of protein-DNA complexes using magnetic tweezers. When a DNA hairpin with multiple binding sites is unzipping, strand separation pauses at the positions bound by a protein. We can thus measure site-specific binding probabilities and dissociation time directly. Taking the TET1 CXXC domain as an example, we show that TET1 CXXC binds multiple CpG motifs with various flanking nucleotides or different methylation patterns in an AT-rich DNA. We are able to establish for the first time, at nanometer resolution, that TET1 CXXC prefers G/C flanked CpG motif over C/G, A/T, or T/A flanked ones. CpG methylation strengthens TET1 CXXC recruitment but has little effect on dissociation time. Finally, we demonstrate that TET1 CXXC can distinguish five CpG clusters in a CpG island with crowded binding motifs. We anticipate that the feasibility of single-molecule multiplexed profiling assays will contribute to the understanding of protein-DNA interactions.

SARS-COV-2 INFECTION AND LONGITUDINAL FECAL SCREENING IN MALAYAN TIGERS (PANTHERA TIGRIS JACKSONI), AMUR TIGERS (PANTHERA TIGRIS ALTAICA ), AND AFRICAN LIONS (PANTHERA LEO KRUGERI) AT THE BRONX ZOO, NEW YORK, USA.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged as the cause of a global pandemic in 2019-2020. In March 2020, New York City became the epicenter in the United States for the pandemic. On 27 March 2020, a Malayan tiger (Panthera tigris jacksoni) at the Bronx Zoo in New York City developed a cough and wheezing with subsequent inappetence. Over the next week, an additional Malayan tiger and two Amur tigers (Panthera tigris altaica) in the same building and three lions (Panthera leo krugeri) in a separate building also became ill. The index case was anesthetized for diagnostic workup. Physical examination and bloodwork results were unremarkable. Thoracic radiography and ultrasonography revealed a bronchial pattern with peribronchial cuffing and mild lung consolidation with alveolar-interstitial syndrome, respectively. SARS-CoV-2 RNA was identified by real-time, reverse transcriptase PCR (rRT-PCR) on oropharyngeal and nasal swabs and tracheal wash fluid. Cytologic examination of tracheal wash fluid revealed necrosis, and viral RNA was detected in necrotic cells by in situ hybridization, confirming virus-associated tissue damage. SARS-CoV-2 was isolated from the tracheal wash fluid of the index case, as well as the feces from one Amur tiger and one lion. Fecal viral RNA shedding was confirmed in all seven clinical cases and an asymptomatic Amur tiger. Respiratory signs abated within 1-5 days for most animals, although they persisted intermittently for 16 days in the index case. Fecal RNA shedding persisted for as long as 35 days beyond cessation of respiratory signs. This case series describes the clinical presentation, diagnostic evaluation, and management of tigers and lions infected with SARS-CoV-2 and describes the duration of viral RNA fecal shedding in these cases. This report documents the first known natural transmission of SARS-CoV-2 from humans to nondomestic felids.

Detection of HPV16/18 E6 Oncoproteins in Head and Neck Squamous Cell Carcinoma Using a Protein Immunochromatographic Assay.

OBJECTIVES/HYPOTHESIS: The accurate diagnostic assessment of clinically relevant human papillomavirus (HPV) infections in patients with head and neck squamous cell carcinoma represents an urgent unmet medical need. The aim of this study was to determine feasibility, accuracy, and clinical significance of HPV16/18 E6 oncoprotein detection on cytological specimens from oropharyngeal squamous cell carcinoma (OPSCC) and neck lymph node metastasis of SCC from unknown primary tumor (CUP) via a protein immunochromatographic assay. STUDY DESIGN: Cross-sectional study. METHODS: Cytological specimens from primary tumor and neck metastases were collected from 34 patients with OPSCC or CUP and applied to a lateral flow format test that detects HPV16 and HPV18 E6 oncoproteins. E6 oncoprotein positivity or negativity in these specimens was compared to the specimens' "HPV-driven" reference status, defined by presence of HPV-DNA in combination with p16INK4a overexpression and/or HPV E6 seropositivity. RESULTS: Eighteen of 29 OPSCC (62%) and three of five CUP (60%) were HPV-driven according to our reference method. The E6 oncoprotein lateral flow test had a sensitivity of 94% (95% CI: 70%-100%) and a specificity of 100% (95% CI: 66%-100%) on primary tumor, and a sensitivity of 88% (95% CI: 64%-99%) and a specificity of 100% (95% CI: 74%-100%) on neck metastases. Test agreement between the E6 lateral flow test and the clinical reference method, HPV-DNA plus p16INK4a was excellent, both for primary lesion and neck metastases. CONCLUSIONS: We found the detection of HPV16/18 E6 oncoproteins to be a feasible, highly reliable, and low-invasive method to assess "HPV-driven" status in OPSCC and CUP. LEVEL OF EVIDENCE: II Laryngoscope, 131:1042-1048, 2021.

Phase separation by ssDNA binding protein controlled via protein-protein and protein-DNA interactions.

Bacterial single-stranded (ss)DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL), and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the Escherichia coli SSB protein forms liquid-liquid phase-separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA, and SSB-interacting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage.

COMET Functions as a PCH2 Cofactor in Regulating the HORMA Domain Protein ASY1.

The formation of the chromosome axis is key to meiotic recombination and hence the correct distribution of chromosomes to meiotic products. A key component of the axis in Arabidopsis is the HORMA domain protein (HORMAD) ASY1, the homolog of Hop1 in yeast and HORMAD1/2 in mammals. The chromosomal association of ASY1 is dynamic, i.e., ASY1 is recruited to the axis at early prophase and later largely removed when homologous chromosomes synapse. PCH2/TRIP13 proteins are well-known regulators of meiotic HORMADs and required for their depletion from synapsed chromosomes. However, no direct interaction has been found between PCH2/TRIP13 and the presumptive HORMAD substrates in any organism other than in budding yeast. Thus, it remains largely elusive how the dynamics of ASY1 and other meiotic HORMADs are controlled. Here, we have identified COMET, the Arabidopsis homolog of human p31comet, which is known for its function in the spindle assembly checkpoint (SAC), as a central regulator of ASY1 dynamics in meiosis. We provide evidence that COMET controls ASY1 localization by serving as an adaptor for PCH2. Because ASY1 accumulates in the cytoplasm in early prophase and is persistently present on chromosomes in comet, we conclude that COMET is required for both the recruitment of ASY1 to the nucleus and the subsequent removal from the axis. The here-revealed function of COMET as an adaptor for PCH2 remarkably resembles the regulation of another HORMAD, Mad2, in the SAC in yeast and animals, revealing a conserved regulatory module of HORMA-domain-containing protein complexes.

The 3D Topography of Mitotic Chromosomes.

A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.

Antibody-based biosensor to detect oncogenic splicing factor Sam68 for the diagnosis of lung cancer.

OBJECTIVE: The present work aimed to investigate the potential utility of Sam68 protein as a prognostic marker in lung cancer. Then an electrochemical immunosensor is fabricated that is sufficiently sensitive to detect Sam68. RESULTS: Analysis of stage-specific Lung cancer microarray data shows that differential expression of Sam68 is associated with cancer stage and monotonically increases from early tumor stage to advanced metastatic stage. Moreover, the higher expression of Sam68 results in reduced survival of lung cancer patients. Based on these observations, an electrochemical immunosensor was developed for the quantification of Sam68 protein. The target protein was captured by the Anti-Sam68 antibody that was immobilized on the modified Glassy carbon electrode. The stepwise assembly process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. This fabricated immunosensor displayed good analytical performance in comparison to commercial ELISA kit with good sensitivity, lower detection limit (LOD) of 10.5 pg mL-1, and wide linear detection range from 1 to 5 µg mL-1. This method was validated with satisfactory detection of Sam68 protein in lung adenocarcinoma cell line, NCI-H23. Besides, spike and recovery assay reconfirm that the sensor can precisely quantify Sam68 protein in a complex physiological sample. CONCLUSION: We conclude Sam68 as a valuable prognostic biomarker for early detection of lung cancer. Moreover, we report the first study on the development of an electrochemical immunosensor for the detection of Sam68. The fabricated immunosensor exhibit excellent analytical performance, which can accurately predict the lung cancer patient pathological state.

Optimized Detection of Protein-Protein and Protein-DNA Interactions, with Particular Application to Plant Telomeres.

Characterization of protein-protein and protein-DNA interactions is critical to understand mechanisms governing the biology of cells. Here we describe optimized methods and their mutual combinations for this purpose: bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), yeast two-hybrid systems (Y2H), and chromatin immunoprecipitation (ChIP). These improved protocols  detect trimeric complexes in which two proteins of interest interact indirectly via a protein sandwiched between them. They also allow isolation of low-abundance chromatin proteins and confirmation that proteins of interest are associated with specific DNA sequences, for example telomeric tracts. Here we describe these methods and their application to map interactions of several telomere- and telomerase-associated proteins and to purify a sufficient amount of chromatin from Arabidopsis thaliana for further investigations (e.g., next-generation sequencing, hybridization).

Dps protein is related to resistance of Mycobacterium abscessus subsp. massiliense against stressful conditions.

Mycobacterium abscessus subsp. massiliense (Mycma) belongs to the Mycobacterium abscessus complex and is a rapidly growing non-tuberculous mycobacterium. The chronic pulmonary, skin, and soft tissue infections that it causes may be difficult to treat due to its intrinsic resistance to the commonly used antimicrobial drugs, making it a serious world public health problem. Iron is an essential nutrient for the growth of microorganisms; nonetheless, it can be toxic when in excess. Thus, bacteria require an iron homeostasis mechanism to succeed in different environments. DNA-binding proteins from starved cells (Dps) are miniferritins with the property to act as additional iron storage proteins but also can bind to DNA, protecting it against hydroxyl radical. Annotation of the Mycma genome revealed the gene mycma_03135 with 79% sequential identity when compared to MSMEG_3242 gene from M. smegmatis mc2 155, which codifies for a known Dps. Recombinant Dps from M. abscessus (rMaDps) was produced in Escherichia coli, purified in soluble form and shown to form high mass oligomers in solution with ferroxidase activity, DNA binding, and protection against damage. The expression of the mycma_03135 gene was induced during Mycma growth in the presence of hydrogen peroxide (H2O2). Additionally, the expression of rMaDps by E. coli conferred greater resistance to H2O2. Thus, this study is the first to identify and characterize a Dps from M. abscessus. KEY POINTS: Mycobacterium abscessus subsp. massiliense express a miniferritin protein (Dps). Mycma Dps binds to DNA and protects against oxidative stress.

Molecular dissection of ALS-linked TDP-43 - involvement of the Gly-rich domain in interaction with G-quadruplex mRNA.

TDP-43 is the major pathogenic protein of amyotrophic lateral sclerosis (ALS). Previously, we identified that TDP-43 interacts with G-quadruplex (G4)-containing RNA and is involved in their long-distance transport in neurons. For the molecular dissection of the TDP-43 and G4-RNA interaction, we analyzed it here in vitro and in cultured cells using a set of 10 mutant TDP-43 proteins from familial and sporadic ALS patients as well as using the TDP-43 C-terminal Gly-rich domain alone. Our results altogether indicate the involvement of the Gly-rich region of TDP-43 in the initial recognition and binding of G4-RNA, which then induces tight binding of TDP-43 with target RNAs, supposedly in conjunction with its RNA recognition motifs.