DNA-binding Domain Protein Research Articles

DNA-Mediated Formation of Phase-Separated Coacervates of the Nucleic Acid-Binding Domain of TAR DNA-Binding Protein (TDP-43) Prevents Its Amyloid-Like Misfolding.

Sequestration of protein molecules and nucleic acids to stress granules is one of the most promising strategies that cells employ to protect themselves from stress. In vitro, studies suggest that the nucleic acid-binding domain of TDP-43 (TDP-43tRRM) undergoes amyloid-like aggregation to β-sheet-rich structures in low pH stress. In contrast, we observed that the TDP-43tRRM undergoes complex coacervation in the presence of ssDNA to a dense and light phase, preventing its amyloid-like aggregation. The soluble light phase consists of monomeric native-like TDP-43tRRM. The microscopic data suggest that the dense phase consists of spherical coacervates with limited internal dynamics. We performed multiparametric analysis by employing various biophysical techniques and found that complex coacervation depends on the concentration and ratio of the participating biomolecules and is driven by multivalent interactions. The modulation of these forces due to environmental conditions or disease mutations regulates the extent of coacervation, and the weakening of interactions between TDP-43tRRM and ssDNA leads to amyloid-like aggregation of TDP-43tRRM. Our results highlight a competition among the native state, amyloid-like aggregates, and complex coacervates tuned by various environmental factors. Together, our results illuminate an alternate function of TDP-43tRRM in response to pH stress in the presence of the ssDNA.

GWAS for Drought Resilience Traits in Red Clover (Trifolium pratense L.)

Red clover (Trifolium pratense L.) is a well-appreciated grassland crop in temperate climates but suffers from increasingly frequent and severe drought periods. Molecular markers for drought resilience (DR) would benefit breeding initiatives for red clover, as would a better understanding of the genes involved in DR. Two previous studies, as follows, have: (1) identified phenotypic DR traits in a diverse set of red clover accessions; and (2) produced genotypic data using a pooled genotyping-by-sequencing (GBS) approach in the same collection. In the present study, we performed genome-wide association studies (GWAS) for DR using the available phenotypic and genotypic data. Single nucleotide polymorphism (SNP) calling was performed using GBS data and the following two red clover genome assemblies: the recent HEN-17 assembly and the Milvus assembly. SNP positions with significant associations were used to delineate flanking regions in both genome assemblies, while functional annotations were retrieved from Medicago truncatula orthologs. GWAS revealed 19 significant SNPs in the HEN-17-derived SNP set, explaining between 5.3 and 23.2% of the phenotypic variation per SNP–trait combination for DR traits. Among the genes in the SNP-flanking regions, we identified candidate genes related to cell wall structuring, genes encoding sugar-modifying proteins, an ureide permease gene, and other genes linked to stress metabolism pathways. GWAS revealed 29 SNPs in the Milvus-derived SNP set that explained substantially more phenotypic variation for DR traits, between 5.3 and 42.3% per SNP–trait combination. Candidate genes included a DEAD-box ATP-dependent RNA helicase gene, a P-loop nucleoside triphosphate hydrolase gene, a Myb/SANT-like DNA-binding domain protein, and an ubiquitin–protein ligase gene. Most accessions in this study are genetically more closely related to the Milvus genotype than to HEN-17, possibly explaining how the Milvus-derived SNP set yielded more robust associations. The Milvus-derived SNP set pinpointed 10 genomic regions that explained more than 25% of the phenotypic variation for DR traits. A possible next step could be the implementation of these SNP markers in practical breeding programs, which would help to improve DR in red clover. Candidate genes could be further characterized in future research to unravel drought stress resilience in red clover in more detail.

Engineering IscB to develop highly efficient miniature editing tools in mammalian cells and embryos

The IscB proteins, as the ancestors of Cas9 endonuclease, hold great promise due to their small size and potential for diverse genome editing. However, their activity in mammalian cells is unsatisfactory. By introducing three residual substitutions in IscB, we observed an average 7.5-fold increase in activity. Through fusing a sequence-non-specific DNA-binding protein domain, the eIscB-D variant achieved higher editing efficiency, with a maximum of 91.3%. Moreover, engineered ωRNA was generated with a 20% reduction in length and slightly increased efficiency. The engineered eIscB-D/eωRNA system showed an average 20.2-fold increase in activity compared with the original IscB. Furthermore, we successfully adapted eIscB-D for highly efficient cytosine and adenine base editing. Notably, eIscB-D is highly active in mouse cell lines and embryos, enabling the efficient generation of disease models through mRNA/ωRNA injection. Our study suggests that these miniature genome-editing tools have great potential for diverse applications.

Impact of C-terminal domains of paralogous single-stranded DNA binding proteins from Streptomyces coelicolor on their biophysical properties and biological functions

Single-stranded DNA-binding proteins (SSB) are crucial in DNA metabolism. While Escherichia coli SSB is extensively studied, the significance of its C-terminal domain has only recently emerged. This study explored the significance of C-domains of two paralogous Ssb proteins in S. coelicolor. Mutational analyses of C-domains uncovered a novel role of SsbA during sporulation-specific cell division and demonstrated that the C-tip is non-essential for survival. In vitro methods revealed altered biophysical and biochemical properties of Ssb proteins with modified C-domains. Determined hydrodynamic properties suggested that the C-domains of SsbA and SsbB occupy a globular position proposed to mediate cooperative binding. Only SsbA was found to form biomolecular condensates independent of the C-tip. Interestingly, the truncated C-domain of SsbA increased the molar enthalpy of unfolding. Additionally, calorimetric titrations revealed that C-domain mutations affected ssDNA binding. Moreover, this analysis showed that the SsbA C-tip aids binding most likely by regulating the position of the flexible C-domain. It also highlighted ssDNA-induced conformational mobility restrictions of all Ssb variants. Finally, the gel mobility shift assay confirmed that the intrinsically disordered linker is essential for cooperative binding of SsbA. These findings highlight the important role of the C-domain in the functioning of SsbA and SsbB proteins.

The CHD family chromatin remodeling enzyme, Kismet, promotes both clathrin-mediated and activity-dependent bulk endocytosis.

Chromodomain helicase DNA binding domain (CHD) proteins, including CHD7 and CHD8, remodel chromatin to enable transcriptional programs. Both proteins are important for proper neural development as heterozygous mutations in Chd7 and Chd8 are causative for CHARGE syndrome and correlated with autism spectrum disorders, respectively. Their roles in mature neurons are poorly understood despite influencing the expression of genes required for cell adhesion, neurotransmission, and synaptic plasticity. The Drosophila homolog of CHD7 and CHD8, Kismet (Kis), promotes neurotransmission, endocytosis, and larval locomotion. Endocytosis is essential in neurons for replenishing synaptic vesicles, maintaining protein localization, and preserving the size and composition of the presynaptic membrane. Several forms of endocytosis have been identified including clathrin-mediated endocytosis, which is coupled with neural activity and is the most prevalent form of synaptic endocytosis, and activity-dependent bulk endocytosis, which occurs during periods of intense stimulation. Kis modulates the expression of gene products involved in endocytosis including promoting shaggy/GSK3β expression while restricting PI3K92E. kis mutants electrophysiologically phenocopy a liquid facets mutant in response to paradigms that induce clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Further, kis mutants do not show further reductions in endocytosis when activity-dependent bulk endocytosis or clathrin-mediated endocytosis are pharmacologically inhibited. We find that Kis is important in postsynaptic muscle for proper endocytosis but the ATPase domain of Kis is dispensable for endocytosis. Collectively, our data indicate that Kis promotes both clathrin-mediated endocytosis and activity-dependent bulk endocytosis possibly by promoting transcription of several endocytic genes and maintaining the size of the synaptic vesicle pool.

In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.

Comparative Structural Investigation of Histone-Like HU Proteins by Small-Angle X-ray Scattering

Nucleoid-associated proteins (NAPs) control the structure and functions of bacterial nucleoid. Histone-like HU proteins are most abundant NAPs in dividing bacterial cells. Previously, structural ensembles of conformations of HU proteins from pathogenic mycoplasmas Spiroplasma melliferum and Mycoplasma gallisepticum were obtained using NMR spectroscopy. A structural study of these mycoplasma proteins is performed by small-angle X-ray scattering (SAXS). The occurrence of individual conformations from the ensemble, obtained by NMR, is estimated from the scattering data on HU protein solutions. In particular, an approach based on characterization of equilibrium mixtures in terms of volume fractions of their components was applied. The general shape of the proteins and their oligomeric state are independently confirmed using ab initio bead modelling. The flexibility of DNA-binding protein domains is analyzed by the ensemble optimization method, which is based on comparison of the structural characteristics of conformations fitting the SAXS data to the distribution of these characteristics in a randomly generated set. The results obtained give a new insight on the variability of the structure of HU proteins, which is necessary for their functioning.

Ahdc1 is a potent regulator of obesity and energy metabolism.

AT-hook DNA-binding motif-containing protein 1 (AHDC1) is a causal gene of intellectual disability/developmental delay in humans. The biological role of AHDC1 is unclear. Recently, some clues from AHDC1 mutation carriers hinted that AHDC1 may participate in body-weight regulation. In this first metabolic phenotype study of Ahdc1 deficiency, we generated a Ahdc1-deficienct mouse line and found that Ahdc1 deficiency in both male and female mice led to adiposity from weaning and obesity characterized by reduced energy expenditure and respiratory quotient, with progressive development of hyperleptinemia, insulin resistance, abnormal glycolipid metabolism, and fatty liver. Our findings show that Ahdc1 is a novel key regulator of obesity and energy metabolism, which provides new insight into the physiological mechanisms of obesity.NEW & NOTEWORTHY In this first metabolic phenotype study of Ahdc1 deficiency, we generated a survivable Ahdc1-deficient mouse line. We found that Ahdc1 deficiency in both male and female mice resulted in adiposity from weaning and obesity characterized by reduced energy expenditure and respiratory quotient. Additionally, there was a progressive development of hyperleptinemia, insulin resistance, abnormal glycolipid metabolism, and fatty liver. These findings demonstrate that Ahdc1 is a novel key regulator of obesity and energy metabolism.

LST-1 is a bifunctional regulator that feeds back on Notch-dependent transcription to regulate C. elegans germline stem cells

Notch signaling regulates stem cells across animal phylogeny. C. elegans Notch signaling activates transcription of two genes, lst-1 and sygl-1, that encode potent regulators of germline stem cells. The LST-1 protein regulates stem cells in two distinct ways: It promotes self-renewal posttranscriptionally and also restricts self-renewal by a poorly understood mechanism. Its self-renewal promoting activity resides in its N-terminal region, while its self-renewal restricting activity resides in its C-terminal region and requires the Zn finger. Here, we report that LST-1 limits self-renewal by down-regulating Notch-dependent transcription. We detect LST-1 in the nucleus, in addition to its previously known cytoplasmic localization. LST-1 lowers nascent transcript levels at both lst-1 and sygl-1 loci but not at let-858, a Notch-independent locus. LST-1 also lowers levels of two key components of the Notch activation complex, the LAG-1 DNA binding protein and Notch intracellular domain (NICD). Genetically, an LST-1 Zn finger mutant increases Notch signaling strength in both gain- and loss-of-function GLP-1/Notch receptor mutants. Biochemically, LST-1 co-immunoprecipitates with LAG-1 from nematode extracts, suggesting a direct effect. LST-1 is thus a bifunctional regulator that coordinates posttranscriptional and transcriptional mechanisms in a single protein. This LST-1 bifunctionality relies on its bipartite protein architecture and is bolstered by generation of two LST-1 isoforms, one specialized for Notch downregulation. A conserved theme from worms to human is the coupling of PUF-mediated RNA repression together with Notch feedback in the same protein.

Finding Novel AMPs Secreted from the Human Microbiome as Potent Antibacterial and Antibiofilm Agents and Studying Their Synergistic Activity with Ag NCs.

Due to the enhanced resistance of bacteria to antibiotics, researchers always try to find effective alternatives to treat drug-resistant bacterial infections. In this context, we have explored antimicrobial peptides (AMPs), which are a broad class of small peptide molecules, and investigated their efficacy as potent antibacterial and antibiofilm agents. AMPs can cause cell death either through disruption of the cell membrane or by inhibiting vital intracellular functions, by binding to RNA, DNA, or intracellular components upon transversion through the cell membrane. We attempted to find potent intracellular cationic AMPs that can demonstrate antibacterial activity through interaction with DNA. As a source of AMPs, we have utilized those that are secreted from the human microbiome with the anticipation that these will be non-toxic in nature. Out of the total 1087 AMPs, 27 were screened on the basis of amino acid length and efficacy to cross the cell membrane barrier. From the list of 27 peptides, 4 candidates were selected through the docking score of these peptides with the DNA binding domain of H2A proteins. Further, the molecular dynamics simulation analysis demonstrated that 2 AMPs, i.e., peptides 7 and 25, are having considerable membrane permeation and DNA binding ability. Further, the in vitro analysis indicated that both peptides 7 and 25 could exhibit potent antibacterial and antibiofilm activities. In order to further enhance the antibiofilm potency, the above AMPs were used as supplements to silver nanoclusters (Ag NCs) to get synergistic activity. The synergistic activity of Ag NCs was found to be significantly increased with both the above AMPs.

Highly Charged Proteins and Their Repulsive Interactions Antagonize Biomolecular Condensation.

Biomolecular condensation is involved in various cellular processes; therefore, regulation of condensation is crucial to prevent deleterious protein aggregation and maintain a stable cellular environment. Recently, a class of highly charged proteins, known as heat-resistant obscure (Hero) proteins, was shown to protect other client proteins from pathological aggregation. However, the molecular mechanisms by which Hero proteins protect other proteins from aggregation remain unknown. In this study, we performed multiscale molecular dynamics (MD) simulations of Hero11, a Hero protein, and the C-terminal low-complexity domain (LCD) of the transactive response DNA-binding protein 43 (TDP-43), a client protein of Hero11, under various conditions to examine their interactions with each other. We found that Hero11 permeates into the condensate formed by the LCD of TDP-43 (TDP-43-LCD) and induces changes in conformation, intermolecular interactions, and dynamics of TDP-43-LCD. We also examined possible Hero11 structures in atomistic and coarse-grained MD simulations and found that Hero11 with a higher fraction of disordered region tends to assemble on the surface of the condensates. Based on the simulation results, we have proposed three possible mechanisms for Hero11's regulatory function: (i) In the dense phase, TDP-43-LCD reduces contact with each other and shows faster diffusion and decondensation due to the repulsive Hero11-Hero11 interactions. (ii) In the dilute phase, the saturation concentration of TDP-43-LCD is increased, and its conformation is relatively more extended and variant, induced by the attractive Hero11-TDP-43-LCD interactions. (iii) Hero11 on the surface of small TDP-43-LCD condensates can contribute to avoiding their fusion due to repulsive interactions. The proposed mechanisms provide new insights into the regulation of biomolecular condensation in cells under various conditions.

A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance.

Fasciola hepatica infection is responsible for substantial economic losses in livestock worldwide and poses a threat to human health in endemic areas. The mainstay of control in livestock and the only drug licenced for use in humans is triclabendazole (TCBZ). TCBZ resistance has been reported on every continent and threatens effective control of fasciolosis in many parts of the world. To date, understanding the genetic mechanisms underlying TCBZ resistance has been limited to studies of candidate genes, based on assumptions of their role in drug action. Taking an alternative approach, we combined a genetic cross with whole-genome sequencing to localise a ~3.2Mbp locus within the 1.2Gbp F. hepatica genome that confers TCBZ resistance. We validated this locus independently using bulk segregant analysis of F. hepatica populations and showed that it is the target of drug selection in the field. We genotyped individual parasites and tracked segregation and reassortment of SNPs to show that TCBZ resistance exhibits Mendelian inheritance and is conferred by a dominant allele. We defined gene content within this locus to pinpoint genes involved in membrane transport, (e.g. ATP-binding cassette family B, ABCB1), transmembrane signalling and signal transduction (e.g. GTP-Ras-adenylyl cyclase and EGF-like protein), DNA/RNA binding and transcriptional regulation (e.g. SANT/Myb-like DNA-binding domain protein) and drug storage and sequestration (e.g. fatty acid binding protein, FABP) as prime candidates for conferring TCBZ resistance. This study constitutes the first experimental cross and genome-wide approach for any heritable trait in F. hepatica and is key to understanding the evolution of drug resistance in Fasciola spp. to inform deployment of efficacious anthelmintic treatments in the field.

TrmB Family Transcription Factor as a Thiol-Based Regulator of Oxidative Stress Response.

ABSTRACTOxidative stress causes cellular damage, including DNA mutations, protein dysfunction, and loss of membrane integrity. Here, we discovered that a TrmB (transcription regulator of mal operon) family protein (Pfam PF01978) composed of a single winged-helix DNA binding domain (InterPro IPR002831) can function as thiol-based transcriptional regulator of oxidative stress response. Using the archaeon Haloferax volcanii as a model system, we demonstrate that the TrmB-like OxsR is important for recovery of cells from hypochlorite stress. OxsR is shown to bind specific regions of genomic DNA, particularly during hypochlorite stress. OxsR-bound intergenic regions were found proximal to oxidative stress operons, including genes associated with thiol relay and low molecular weight thiol biosynthesis. Further analysis of a subset of these sites revealed OxsR to function during hypochlorite stress as a transcriptional activator and repressor. OxsR was shown to require a conserved cysteine (C24) for function and to use a CG-rich motif upstream of conserved BRE/TATA box promoter elements for transcriptional activation. Protein modeling suggested the C24 is located at a homodimer interface formed by antiparallel α helices, and that oxidation of this cysteine would result in the formation of an intersubunit disulfide bond. This covalent linkage may promote stabilization of an OxsR homodimer with the enhanced DNA binding properties observed in the presence of hypochlorite stress. The phylogenetic distribution TrmB family proteins, like OxsR, that have a single winged-helix DNA binding domain and conserved cysteine residue suggests this type of redox signaling mechanism is widespread in Archaea.

Effect of icaritin on autophagy-related protein expression in TDP-43-transfected SH-SY5Y cells.

ObjectiveTo study the protective effect and mechanism of icaritin (ICT) in a SH-SY5Y cells with virus-loaded TAR DNA-binding domain protein 43(TDP-43) by examining the effect of ICT on the expression of autophagy-related proteins in TDP-43-infected SH-SY5Y cells.MethodsA TDP-43-induced neuronal cell injury model was established by transfecting well-growing SH-SY5Y cells with virus loaded with the TDP-43 gene. The changes in cell viability were detected by the CCK-8 method. After successful transfection, the establishment of the model was verified by real-time quantitative PCR (qPCR) and Western blot methods. After the cells were subjected to drug intervention with ICT, the changes in the expression levels of TDP-43, cleaved Caspase-3, LC3 II/I, Beclin-1 and p62 were detected by Western blotting.ResultsAfter ICT intervention, it was found that compared with that of the TDP-43 group, the cell viability of the TDP-43+ICT group increased, the expression level of TDP-43 decreased, and the expression levels of the apoptotic protein cleaved Caspase-3, autophagy protein Beclin-1, and LC3-II/I decreased, while the expression level of the autophagy protein p62 increased.ConclusionICT has a protective effect on the SH-SY5Y cell injury model transfected with TDP-43. This protective effect may be related to reducing the protein expression of TDP-43 and inhibiting autophagy.

Quantum technology a tool for sequencing of the ratio DSS/DNA modifications for the development of new DNA-binding proteins

ABSTRACT The current study summarizes the literature on quantum-based structural sequencing methods and their predicted roles in the discovery of new DNA-binding protein domains. Although there is no literature yet showing the role of quantum structural sequencing for the discovery of new domains for DNA-binding proteins, the current study shows that the development of new quantum-based and far-from-equilibrium biological systems can be achieved by 3D-curved room-temperature superconductivity materials. The technology is useful for protein engineering and 3D sequencing of the ratio DNA/RNA secondary structures (DSS)/Physiological Modifications (PM) and will overcome the use of nanopore and ChiP sequencing. This approach is very useful for the protein engineering of recombinant enzymes for the unnatural replication of proteins and glycans for new biochemical activities, treatment of HIV and cancers, new genome editing machinery, artificial replication and translation, first sequencing of DSS/physiological modification/telomere with a correlation to cancer genesis and therapy, The key DSS in this study are: quadruplex, hairpin loop, triplexes and duplexes, i-motif, Holliday junction; and the PM investigated: 6 mA, 6fmA, 5mC, 5fC, 8-oxo-guanine. The DSS/PM ratio is critical for proto-oncogene activation and cancer genesis.

Interaction Analysis between the Arabidopsis Transcription Repressor VAL1 and Transcription Coregulators SIN3-LIKEs (SNLs).

VIVIPAROUS1/ABSCISIC ACID INSENSITIVE3-LIKE1 (VAL1) encodes a DNA-binding B3 domain protein and plays essential roles in seed maturation and flowering transition by repressing genes through epigenetic silencing in Arabidopsis. SWI-INDEPENDENT3 (SIN3)-LIKEs (SNLs), which encode scaffold proteins for the assembly of histone deacetylase complexes and have six SIN3 homologues (SNL1–SNL6) in Arabidopsis thaliana, directly repress gene expression to regulate seed maturation and flowering transition. However, it remains unclear whether VAL1 and SNLs work together in repressing the expression of related genes. In this study, yeast two-hybrid and firefly luciferase complementation imaging assays revealed that VAL1 interacts with SNLs, which can be attributed to its own zinc-finger CW (conserved Cys (C) and Trp (W) residues) domain and the PAH (Paired Amphipathic Helices) domains of SNLs. Furthermore, pull-down experiments confirmed that the CW domain of VAL1 interacts with both intact protein and the PAH domains of SNLs proteins, and the co-immunoprecipitation assays also confirmed the interaction between VAL1 and SNLs. In addition, quantitative real-time PCR (qRT-PCR) analysis showed that VAL1 and SNLs were expressed in seedlings, and transient expression assays showed that VAL1 and SNLs were localized in the nucleus. Considered together, these results reveal that VAL1 physically interacts with SNLs both in vitro and in vivo, and suggest that VAL1 and SNLs may work together to repress the expression of genes related to seed maturation and flowering transition in Arabidopsis.

The DNA-Binding High-Mobility Group Box Domain of Sox Family Proteins Directly Interacts with RNA In Vitro.

There is a growing body of evidence that a substantial number of protein domains identified as DNA-binding also interact with RNA to regulate biological processes. Several recent studies have revealed that the Sox2 transcription factor binds RNA through its high-mobility group box (HMGB) domain in vitro and in vivo. A high degree of conservation of this domain among members of the Sox family of transcription factors suggests that RNA-binding activity may be a general feature of these proteins. To address this hypothesis, we examined a subset of HMGB domains from human Sox family of proteins for their ability to bind both DNA and RNA in vitro. We observed selective, high-affinity interactions between Sox family HMGB domains and various model RNA elements, including a four-way junction RNA, a hairpin RNA with an internal bulge, G-quadruplex RNA, and a fragment of long noncoding RNA ES2, which is known to directly interact with Sox2. Importantly, the HMGB domains bind these RNA ligands significantly tighter than nonconsensus dsDNA and in some cases with affinities rivaling those of their consensus dsDNA sequences. These data suggest that RNA binding is a conserved feature of the Sox family of transcription factors with the potential to modulate unappreciated biological functions.

Investigating the interaction of E6/E6AP heterodimer with p53‐DBD by fluorescence resonance energy transfer

Human papilloma viruses (HPV) are small DNA viruses, among which the type 16 is the most prevalent and responsible for about 50% of human cervical carcinomas. The E6 oncoprotein of HPV‐16 interacts with numerous host proteins by recognizing their LXXLL motifs. One of the HPV‐16 E6 cellular binding targets is the E3 ubiquitin ligase E6AP. Once binding, the E6/E6AP heterodimer recruits and ubiquitinates p53, leading to subsequent degradation of p53. Importantly, neither E6 nor E6AP interacts with p53 alone. Therefore, blocking the interaction between E6 and E6AP could stabilize p53. Indeed, a LQELL peptide has been found capable to interact with E6 and suppress degradation of p53. Interaction of ligands with p53 may also reduce or inhibit binding of p53 with E6. While the crystal structure of E6/E6AP/p53 has been determined, efficient ligands targeting E6/E6AP/p53 are still missing. In this work, we studied the interactions between E6/E6AP fusion protein and p53 DNA binding domain using fluorescence resonance energy transfer (FRET). E6/E6AP fused to YFP and p53 fused to CFP were expressed from E. coli and purified using Ni affinity chromatography. We characterized the complex stability and obtained a dissociation constant consistent with previously determined by isothermal titration calorimetry. We further obtained the rate constant for dissociation of p53 from E6/E6AP using unlabeled p53 protein. Since the intensity of FRET signal is related to the amount of E6/E6AP/p53 complex, we further investigated the effect of p28, a p53 binding peptide, on the stability of E6/E6AP/p53 complex. Our preliminary results showed that p28 interferes with the binding of p53 to E6/E6AP.

Watching liquid droplets of TDP-43CTD age by Raman spectroscopy

Liquid–liquid phase separation (LLPS) is a biological phenomenon wherein a metastable and concentrated droplet phase of biomolecules spontaneously forms. A link may exist between LLPS of proteins and the disease-related process of amyloid fibril formation; however, this connection is not fully understood. Here, we investigated the relationship between LLPS and aggregation of the C-terminal domain of TAR DNA-binding protein 43, an amyotrophic lateral sclerosis–related protein known to both phase separate and form amyloids, by monitoring conformational changes during droplet aging using Raman spectroscopy. We found that the earliest aggregation events occurred within droplets as indicated by the development of β-sheet structure and increased thioflavin-T emission. Interestingly, filamentous aggregates appeared outside the solidified droplets at a later time, suggestive that amyloid formation is a heterogeneous process under LLPS solution conditions. Furthermore, the secondary structure content of aggregated structures inside droplets is distinct from that in de novo fibrils, implying that fibril polymorphism develops as a result of different environments (LLPS versus bulk solution), which may have pathological significance.

Genetic Variability of the Functional Domains of Chromodomains Helicase DNA-Binding (CHD) Proteins.

In the past few years, there has been an increasing neuroscientific interest in understanding the function of mammalian chromodomains helicase DNA-binding (CHD) proteins due to their association with severe developmental syndromes. Mammalian CHDs include nine members (CHD1 to CHD9), grouped into subfamilies according to the presence of specific functional domains, generally highly conserved in evolutionary terms. Mutations affecting these domains hold great potential to disrupt protein function, leading to meaningful pathogenic scenarios, such as embryonic defects incompatible with life. Here, we analysed the evolution of CHD proteins by performing a comparative study of the functional domains of CHD proteins between orthologous and paralogous protein sequences. Our findings show that the highest degree of inter-species conservation was observed at Group II (CHD3, CHD4, and CHD5) and that most of the pathological variations documented in humans involve amino acid residues that are conserved not only between species but also between paralogs. The parallel analysis of both orthologous and paralogous proteins, in cases where gene duplications have occurred, provided extra information showing patterns of flexibility as well as interchangeability between amino acid positions. This added complexity needs to be considered when the impact of novel mutations is assessed in terms of evolutionary conservation.