Acidic Domain Research Articles

Disordered regions and folded modules in CAF-1 promote histone deposition in Schizosaccharomyces pombe.

Genome and epigenome integrity in eukaryotes depends on the proper coupling of histone deposition with DNA synthesis. This process relies on the evolutionary conserved histone chaperone CAF-1 for which the links between structure and functions are still a puzzle. While studies of the Saccharomyces cerevisiae CAF-1 complex enabled to propose a model for the histone deposition mechanism, we still lack a framework to demonstrate its generality and in particular, how its interaction with the polymerase accessory factor PCNA is operating. Here, we reconstituted a complete SpCAF-1 from fission yeast. We characterized its dynamic structure using NMR, SAXS and molecular modeling together with in vitro and in vivo functional studies on rationally designed interaction mutants. Importantly, we identify the unfolded nature of the acidic domain which folds up when binding to histones. We also show how the long KER helix mediates DNA binding and stimulates SpCAF-1 association with PCNA. Our study highlights how the organization of CAF-1 comprising both disordered regions and folded modules enables the dynamics of multiple interactions to promote synthesis-coupled histone deposition essential for its DNA replication, heterochromatin maintenance, and genome stability functions.

Molecular switching in transcription through splicing and proline-isomerization regulates stress responses in plants

The Arabidopsis thaliana DREB2A transcription factor interacts with the negative regulator RCD1 and the ACID domain of subunit 25 of the transcriptional co-regulator mediator (Med25) to integrate stress signals for gene expression, with elusive molecular interplay. Using biophysical and structural analyses together with high-throughput screening, we reveal a bivalent binding switch in DREB2A containing an ACID-binding motif (ABS) and the known RCD1-binding motif (RIM). The RIM is lacking in a stress-induced DREB2A splice variant with retained transcriptional activity. ABS and RIM bind to separate sites on Med25-ACID, and NMR analyses show a structurally heterogeneous complex deriving from a DREB2A-ABS proline residue populating cis- and trans-isomers with remote impact on the RIM. The cis-isomer stabilizes an α-helix, while the trans-isomer may introduce energetic frustration facilitating rapid exchange between activators and repressors. Thus, DREB2A uses a post-transcriptionally and post-translationally modulated switch for transcriptional regulation.

Osteocalcin: Promoter or Inhibitor of Hydroxyapatite Growth?

Osteocalcin is the most abundant noncollagenous bone protein and the functions in bone remineralization as well as in inhibition of bone growth have remained unclear. In this contribution, we explain the dual role of osteocalcin in the nucleation of new calcium phosphate during bone remodeling and in the inhibition of hydroxyapatite crystal growth at the molecular scale. The mechanism was derived using pH-resolved all-atom models for the protein, phosphate species, and hydroxyapatite, along with molecular dynamics simulations and experimental and clinical observations. Osteocalcin binds to (hkl) hydroxyapatite surfaces through multiple residues, identified in this work, and the fingerprint of binding residues varies as a function of the (hkl) crystal facet and pH value. On balance, the affinity of osteocalcin to hydroxyapatite slows down crystal growth. The unique tricalcium γ-carboxylglutamic acid (Gla) domain hereby rarely adsorbs to hydroxyapatite surfaces and faces instead toward the solution. The Gla domain enables prenucleation of calcium phosphate for new bone formation at a slightly acidic pH of 5. The growth of prenucleation clusters of calcium phosphate continues upon increase in pH value from 5 to 7 and is much less favorable, or not observed, on the native osteocalcin structure at and above neutral pH values of 7. The results provide mechanistic insight into the early stages of bone remodeling from the molecular scale, help inform mutations of osteocalcin to modify binding to apatites, support drug design, and guide toward potential cures for osteoporosis and hyperosteogeny.

Clinico-bacteriological Study and Molecular Detection of Campylobacter in Childhood Diarrhoea: A Cross-sectional Study

Introduction: Campylobacter infections cause diarrhoeal diseases as frequently as Salmonella and Shigella infections. The prevalence of Campylobacter infection among children with acute diarrhoea in developing countries ranges from 5-35%. Diagnosing Campylobacter infections is challenging as the organism is difficult to isolate, grow, and identify. Currently, no best-practice clinical or public health laboratory guidelines exist for laboratory diagnosis of Campylobacter infections. Aim: To explore the clinical and bacterial aspects of childhood diarrhoea, emphasising the prevalence and molecular detection of Campylobacter. Materials and Methods: A hospital-based cross-sectional descriptive study was conducted with 55 stool samples of children under five with diarrhoea or dysentery at the Department of Microbiology, JSS Medical College, Mysuru, Karnataka, India, from October 2016 to September 2017. All stool samples were inoculated onto Campylobacter selective and non selective media with filtration and incubated in microaerophilic conditions. The culture isolates were identified by standard phenotypic tests. Molecular characterisation of Campylobacter was performed targeting the Campylobacter adhesion to fibronectin F (cadF) gene. The presence of a phylogenetically conserved 16S ribosomal Ribonucleic Acid (16SrRNA) domain was studied, followed by specific detection of pathogenic Campylobacter species. The Statistical Package for Social Sciences (SPSS) version 22.0 was used for statistical analysis. Descriptive statistics like percentage, mean, and Standard Deviation (SD) were applied. Results: Campylobacter was isolated by culture in one out of 55 stool samples. The isolate was confirmed to be Campylobacter jejuni by phenotypic tests. Campylobacter genus-level Polymerase Chain Reaction (PCR) was positive for 6 samples (10.9%). Six positive samples were subjected to species-level PCR; all were positive for C. jejuni. Out of 55 stool samples, two diarrheagenic Escherichia coli, two Shigella sonnei, one Shigella dysenteriae, and one Salmonella enterica serovar Typhi were also identified. Conclusion: Culture is insufficiently sensitive for diagnosing Campylobacter infection compared with nucleic acid-based diagnostics. Nucleic acid-based diagnostics offer increased sensitivity, can determine both the presence and burden of infection, and can distinguish between Campylobacter infections at the species level. Therefore, PCR is recommended, if feasible, as the preferred diagnostic modality for detecting Campylobacter infection.

Capturing Protein-Protein Interactions with Acidic Amino Acids Reactive Cross-Linkers.

Acidic residues (Asp and Glu) have a high prevalence on protein surfaces, but cross-linking reactions targeting these residues are limited. Existing methods either require high-concentration coupling reagents or have low structural compatibility. Here a previously reported "plant-and-cast" strategy is extended to develop heterobifunctional cross-linkers. These cross-linkers first react rapidly with Lys sidechains and then react with Asp and Glu sidechains, in a proximity-enhanced fashion. The cross-linking reaction proceeds at neutral pH and room temperature without coupling reagents. The efficiency and robustness of cross-linking using model proteins, ranging from small monomeric proteins to large protein complexes are demonstrated. Importantly, it is shown that this type of cross-linkers are efficient at identifying protein-protein interactions involving acidic domains. The Cross-linking mass spectrometry (XL-MS) study with p53 identified 87 putative binders of the C-terminal domain of p53. Among them, SARNP, ZRAB2, and WBP11 are shown to regulate the expression and alternative splicing of p53 target genes. Thus, these carboxylate-reactive cross-linkers will further expand the power of XL-MS in the analysis of protein structures and protein-protein interactions.

The Stilbene Synthase Family in Arachis: A Genome-Wide Study and Functional Characterization in Response to Stress.

Peanut (Arachis hypogaea) and its wild relatives are among the few species that naturally synthesize resveratrol, a well-known stilbenoid phytoalexin that plays a crucial role in plant defense against biotic and abiotic stresses. Resveratrol has received considerable attention due to its health benefits, such as preventing and treating various human diseases and disorders. Chalcone (CHS) and Stilbene (STS) Synthases are plant-specific type III Polyketide Synthases (PKSs) that share the same substrates and are key branch enzymes in the biosynthesis of flavonoids and stilbenoids, respectively. Although resveratrol accumulation in response to external stimulus has been described in peanut, there are no comprehensive studies of the CHS and STS gene families in the genus Arachis. In the present study, we identified and characterized 6 CHS and 46 STS genes in the tetraploid peanut and an average of 4 CHS and 22 STS genes in three diploid wild species (Arachis duranensis, Arachis ipaënsis and Arachis stenosperma). The CHS and STS gene and protein structures, chromosomal distributions, phylogenetic relationships, conserved amino acid domains, and cis-acting elements in the promoter regions were described for all Arachis species studied. Based on gene expression patterns of wild A. stenosperma STS genes in response to different biotic and abiotic stresses, we selected the candidate AsSTS4 gene, which is strongly induced by ultraviolet (UV) light exposure, for further functional investigation. The AsSTS4 overexpression in peanut hairy roots significantly reduced (47%) root-knot nematode infection, confirming that stilbene synthesis activation in transgenic plants can increase resistance to pathogens. These findings contribute to understanding the role of resveratrol in stress responses in Arachis species and provide the basis for genetic engineering for improved production of valuable secondary metabolites in plants.

Abstract A178: Therapeutic potential of a p14ARF minimal domain peptide in non-small cell lung cancer

Abstract Many current therapeutic efforts in cancer biology focus on inhibiting the function of aberrant oncogenes whose activities are known to increase proliferation and cell growth. Unfortunately, these efforts have had limited success in lung cancer, where the five-year survival rate remains unchanged. We sought a novel approach; rather than inhibiting oncogenes, we worked to reestablish the function of the p14ARF tumor suppressor in non-small cell lung cancer (NSCLC) to block tumor growth and proliferation. The p14ARF tumor suppressor is encoded by the CDKN2A gene and is the second most commonly mutated gene in NSCLC. p14ARF is known to play a fundamental role in preventing cancer progression by regulating senescence, apoptosis, and proliferation. Here, to determine the minimal functional domain of p14ARF, we generated a panel of p14ARF expression mutants, deleting various amino acids within the known functional and conserved domains of p14ARF. These mutants were then evaluated for their ability to function as wild type p14ARF. Through proliferative and transformative studies, we identified a 10-amino acid domain that is required for p14ARF function. New cell-penetrating peptide technology was employed to determine if these resides were sufficient to elicit antiproliferative effects on their own. We now show that this small region of ARF is capable of entering tumor cells when linked to a cell penetrating peptide. Moreover, this small cell penetrating ARF peptide retains all of ARF’s known tumor suppressor functions. Our findings provide evidence that small cell penetrating peptides are capable of inhibiting tumor cell growth and proliferation. These experiments also lay the foundation for more precise therapeutic peptide cancer treatments, ones that could be used to restore p14ARF tumor suppressor function. Citation Format: Anna L Grobelny, Leonard B Maggi, Jason D Weber. Therapeutic potential of a p14ARF minimal domain peptide in non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A178.

Abstract 14233: Checkpoint Kinase 1 Combines PKM2 C-domain to Stimulate Endogenous Cardiomyocyte Renewal and Cardiac Repair by Activating Cardiac Metabolic Reprogramming in a Porcine Model of Myocardial Ischemia/Reperfusion Injury

Background: The regenerative capacity of adult mammalian hearts is limited. We previously reported CHK1 in adult mice could promote cardiac regeneration after MI. However, the specific mechanism of CHK1 needs more comprehensive and in-depth interpretation. Methods: I/R injury was induced in pigs, recombinant human checkpoint kinase 1 (rhCHK1) protein encapsulated in hydrogel was injected into the infarct border zone. The therapeutic efficacy of rhCHK1 was evaluated by echocardiography, cMRI, hematological indices and immunofluorescence. Multi-omics analysis were performed to explore the potential mechanisms of rhCHK1. Further, protein interaction prediction and CO-IP experiments were conducted to verify the specific interaction pattern and domains between CHK1 and PKM2. Hipsc-CMs and porcine were used for confirming whether rhCHK1 promotes cardiac regeneration through combining with the PKM2 C-domain. Results: RhCHK1 protein stimulated CMs proliferation and reduced cardiac inflammation at 3 dpi, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 dpi. Mechanistically, multiomics analysis showed that glycolysis and mTOR pathways were significantly enriched in this process. Further CO-IP experiments and protein docking prediction showed that CHK1 directly bound to and activated the S37 and Y105 sites of PKM2 to promote metabolic reprogramming. We further constructed plasmids that knock out different CHK1 and PKM2 domains, and transfected them into H293T cells for CO-IP experiments. It was found that the 391-476 domain of CHK1 directly binds to the 157-400 amino acids of PKM2. Further hipsc-CMs and pig experiments both demonstrated that CHK1 stimulates CMs renewal and cardiac repair by activating PKM2 C-domain-mediated cardiac metabolic reprogramming and mTORC1 pathway. Conclusion: This study demonstrates that rhCHK1 could combine with PKM2 391-476 amino acid domain to mediate metabolic reprogramming and mTORC1 pathways to promote CM proliferation and myocardial repair. This study is the first to clarify the specific functional mechanism of CHK1 in pigs. Our results underscore the potential of the local applied rhCHK1 as a safe and simple feasible treatment option for repairing heart.

Mechanism of synergistic activation of Arp2/3 complex by cortactin and WASP-family proteins

Cortactin coactivates Arp2/3 complex synergistically with WASP-family nucleation-promoting factors (NPFs) and stabilizes branched networks by linking Arp2/3 complex to F-actin. It is poorly understood how cortactin performs these functions. We describe the 2.89 Å resolution cryo-EM structure of cortactin’s N-terminal domain (Cort1-76) bound to Arp2/3 complex. Cortactin binds Arp2/3 complex through an inverted Acidic domain (D20-V29), which targets the same site on Arp3 as the Acidic domain of NPFs but with opposite polarity. Sequences N- and C-terminal to cortactin’s Acidic domain do not increase its affinity for Arp2/3 complex but contribute toward coactivation with NPFs. Coactivation further increases with NPF dimerization and for longer cortactin constructs with stronger binding to F-actin. The results suggest that cortactin contributes to Arp2/3 complex coactivation with NPFs in two ways, by helping recruit the complex to F-actin and by stabilizing the short-pitch (active) conformation, which are both byproducts of cortactin’s core function in branch stabilization.

Tuning aldol condensation product selectivity by controlling diffusion and reaction rates in zeolites

This study presents a method for quantifying mass-transfer limitations in microporous acid zeolites that combines a titration technique and a reaction–diffusion model. The method involves partially exchanging H+ sites with Na+ sites to adjust the site density and observing the resulting reaction rates for the aldol condensation of acetone and cyclopentanone. When the intrinsic activity per site is uniform throughout the zeolite and in the absence of mass-transport limitations, a linear decrease in the reaction rate is expected as the density of sites is systematically lowered by gradual titration. Conversely, if mass-transport limitations are present, the initial decrease in rate with increasing number of titrated sites is much lower than that observed when a large fraction of the active sites have been titrated. This non-linear variation of observed rate as a function of the density of titrated sites indicates the presence of mass-transport limitations in the system. Near the limit where the density of remaining active sites is low enough to avoid generating a concentration gradient inside the zeolite crystallite, the variation in rate becomes linear. The titration results can be coupled with reaction–diffusion modeling to fit and calculate the Thiele modulus, which provides quantitative information on the extent of mass-transport limitations in the studied porous acidic domains. In the case of acetone-cyclopentanone cross-condensation reaction, the selectivity towards the different products was found to be influenced by the degree of mass transfer limitations. That is, kinetically driven systems favor the cyclopentanone self-condensation product while diffusion-limited environments favor products of the reactant with faster diffusion rates. Accordingly, the manipulation of site density was found to be useful in tuning product selectivity towards specific desired products, which demonstrates the potential of this method for developing and optimizing microporous catalysts with controllable acid site density and provide valuable insight into the relationship between mass-transfer limitations and product selectivity.

Expression pattern analysis and characterization of the hereditary sensory and autonomic neuropathy 2 A (HSAN2A) gene with no lysine kinase (WNK1) in human dorsal root ganglion

Inherited painless neuropathies arise due to genetic insults that either block the normal signaling of or destroy the sensory afferent neurons in the dorsal root ganglion (DRG) responsible for transducing noxious stimuli. Complete loss of these neurons leads to profound insensitivity to all sensory modalities including pain. Hereditary sensory and autonomic neuropathy type 2 (HSNAII) is a rare genetic neuropathy characterized by a progressive distal early onset sensory loss. This syndrome is caused by autosomal recessive mutations in the with-no-lysine protein kinase 1 (WNK1) serine-threonine kinase gene. Of interest, disease-associated mutations are found in the large exon, termed “HSN2,” which encodes a 498 amino acid domain C-terminal to the kinase domain. These mutations lead to truncation of the HSN2-containing proteins through the addition of an early stop codon (nonsense mutation) leading to loss of the C-terminal domains of this large protein. The present study evaluates the transcripts, gene structure, and protein structure of HSN2-containing WNK1 splice variants in DRG and spinal cord in order to establish the basal expression patterns of WNK1 and HSN2-containing WNK1 splice variants using multiplex fluorescent situ hybridization. We hypothesized that these transcripts would be enriched in pain-sensing DRG neurons, and, potentially, that enrichment in nociceptive neurons was responsible for the painless phenotypes observed. However, our in-depth analyses revealed that the HSN2-WNK1 splice variants were ubiquitously expressed but were not enriched in tachykinin 1-expressing C-fiber neurons, a class of neurons with a highly nociceptive character. We subsequently identified other subpopulations of DRG neurons with higher levels of HSN2-WNK1 expression, including mechanosensory large fibers. These data are inconsistent with the hypothesis that this transcript is enriched in nociceptive fibers, and instead suggest it may be related to general axon maintenance, or that nociceptive fibers are more sensitive to the genetic insult. These findings clarify the molecular and cellular expression pattern of this painless neuropathy gene in human tissue.

Monovalent Salt and pH-Stimulated Gelation of Scallop (Patinopecten yessoensis) Male Gonad Hydrolysates/κ-Carrageenan.

The gelation of scallop Patinopecten yessoensis male gonad hydrolysates (SMGHs) and κ-carrageenan (KC) subjected to pH (2-8, 3-9) and NaCl/KCl stimuli-response was investigated. SMGHs/KC gels subjected to a NaCl response exhibited an increasing storage modulus G'from 2028.6 to 3418.4 Pa as the pH decreased from pH 8 to 2, with corresponding T23 fluctuating from 966.40 to 365.64 ms. For the KCl-treated group, SMGHs/KC gels showed an even greater G' from 4646.7 to 10996.5 Pa, with T23 fluctuating from 622.2 to 276.98 ms as the pH decreased from 9 to 3. The improved gel strength could be ascribed to the blueshift and redshift of hydroxyl groups and amide I peaks, enhanced enthalpy and peak temperature, and gathered characteristic diffraction peaks from SMGHs, KC, NaCl, and KCl. The CLSM and cryo-SEM images further reflected that SMGHs/KC gels showed more flocculation formation and denser and more homogeneous networks with smaller pore sizes in more acidic domains, especially when subjected to the KCl response. This research gives a theoretical and methodological understanding of the construction of salt- and pH-responsive SMGHs/KC hydrogels as novel functional soft biomaterials applied in food and biological fields.

A Spanish Family with Gordon Syndrome Due to a Variant in the Acidic Motif of WNK1.

(1) Background: Gordon syndrome (GS) or familial hyperkalemic hypertension is caused by pathogenic variants in the genes WNK1, WNK4, KLHL3, and CUL3. Patients presented with hypertension, hyperkalemia despite average glomerular filtration rate, hyperchloremic metabolic acidosis, and suppressed plasma renin (PR) activity with normal plasma aldosterone (PA) and sometimes failure to thrive. GS is a heterogeneous genetic syndrome, ranging from severe cases in childhood to mild and sometimes asymptomatic cases in mid-adulthood. (2) Methods: We report here a sizeable Spanish family of six patients (four adults and two children) with GS. (3) Results: They carry a novel heterozygous missense variant in exon 7 of WNK1 (p.Glu630Gly). The clinical presentation in the four adults consisted of hypertension (superimposed pre-eclampsia in two cases), hyperkalemia, short stature with low body weight, and isolated hyperkalemia in both children. All patients also presented mild hyperchloremic metabolic acidosis and low PR activity with normal PA levels. Abnormal laboratory findings and hypertension were normalized by dietary salt restriction and low doses of thiazide or indapamide retard. (4) Conclusions: This is the first Spanish family with GS with a novel heterozygous missense variant in WNK1 (p.Glu630Gly) in the region containing the highly conserved acidic motif, which is showing a relatively mild phenotype, and adults diagnosed in mild adulthood. These data support the importance of missense variants in the WNK1 acidic domain in electrolyte balance/metabolism. In addition, findings in this family also suggest that indapamide retard or thiazide may be an adequate long-standing treatment for GS.

Crystal structure of histone deacetylase 6 complexed with (R)-lipoic acid, an essential cofactor in central carbon metabolism

The enzyme cofactor (R)-lipoic acid plays a critical role in central carbon metabolism due to its catalytic function in the generation of acetyl-CoA, which links glycolysis with the tricarboxylic acid cycle. This cofactor is also essential for the generation of succinyl CoA within the tricarboxylic acid cycle. However, the biological functions of (R)-lipoic acid extend beyond metabolism owing to its facile redox chemistry. Most recently, the reduced form of (R)-lipoic acid, (R)-dihydrolipoic acid, has been shown to inhibit histone deacetylases (HDACs) with selectivity for the inhibition of HDAC6. Here, we report the 2.4 Å-resolution X-ray crystal structure of the complex between (R)-dihydrolipoic acid and HDAC6 catalytic domain 2 from Danio rerio, and we report a dissociation constant (KD) of 350nM for this complex as determined by isothermal titration calorimetry. The crystal structure illuminates key affinity determinants in the enzyme active site, including thiolate-Zn2+ coordination and S-π interactions in the F583-F643 aromatic crevice. This study provides the first visualization of the connection between HDAC function and the biological response to oxidative stress: the dithiol moiety of (R)-dihydrolipoic acid can serve as a redox-regulated pharmacophore capable of simultaneously targeting the catalytic Zn2+ ion and the aromatic crevice in the active site of HDAC6.

Structural insights into histone binding and nucleosome assembly by chromatin assembly factor-1.

Chromatin inheritance entails de novo nucleosome assembly after DNA replication by chromatin assembly factor-1 (CAF-1). Yet direct knowledge about CAF-1's histone binding mode and nucleosome assembly process is lacking. In this work, we report the crystal structure of human CAF-1 in the absence of histones and the cryo-electron microscopy structure of CAF-1 in complex with histones H3 and H4. One histone H3-H4 heterodimer is bound by one CAF-1 complex mainly through the p60 subunit and the acidic domain of the p150 subunit. We also observed a dimeric CAF-1-H3-H4 supercomplex in which two H3-H4 heterodimers are poised for tetramer assembly and discovered that CAF-1 facilitates right-handed DNA wrapping of H3-H4 tetramers. These findings signify the involvement of DNA in H3-H4 tetramer formation and suggest a right-handed nucleosome precursor in chromatin replication.

Characterization of a crustin-like peptide involved in shrimp immune response to bacteria and Enterocytozoon hepatopenaei (EHP) infection in Palaemon carinicauda.

Crustins represent one type of antimicrobial peptides (AMPs) that are key components of the innate immune process of crustaceans. This study successfully identified a novel crustin-like peptide, EcCrustin2, in ridgetail white prawn, Palaemon carinicauda (formerly Exopalaemon carinicauda). EcCrustin2 was found to be 1082 bp in length, with a 378 bp open reading frame (ORF) encoding 125 amino acids. The deduced amino acid sequence of EcCrustin2 exhibited characteristics of crustins in crustacean, including a Cys-rich region at the N-terminus as well as a whey acidic protein domain at the C-terminus. Phylogenetic analysis revealed that the EcCrustin2 was first clustered with Type I crustins, then with other crustins. Expression of EcCrustin2 was mainly detected in immune tissues, including hemocytes, gill and stomach. The expression level of EcCrustin2 was also significantly up-regulated after being exposed to lipopolysaccharide (LPS), lipoteichoic acid (LTA), Vibrio parahaemolyticus and Staphylococcus aureus. EHP infection could also induce EcCrustin2 expression in P. carinicauda. Knockdown of EcCrustin2 with siRNA increased the mortality of V. parahaemolyticus challenged shrimp. Finally, the recombinant EcCrustin2 protein was obtained and demonstrated a wide spectrum of antibacterial activity in vitro. These results indicated that EcCrustin2 takes part in the immune response against bacteria and EHP infection.

Clusters of acidic and hydrophobic residues can predict acidic transcriptional activation domains from protein sequence.

Transcription factors activate gene expression in development, homeostasis, and stress with DNA binding domains and activation domains. Although there exist excellent computational models for predicting DNA binding domains from protein sequence, models for predicting activation domains from protein sequence have lagged, particularly in metazoans. We recently developed a simple and accurate predictor of acidic activation domains on human transcription factors. Here, we show how the accuracy of this human predictor arises from the clustering of aromatic, leucine, and acidic residues, which together are necessary for acidic activation domain function. When we combine our predictor with the predictions of convolutional neural network (CNN) models trained in yeast, the intersection is more accurate than individual models, emphasizing that each approach carries orthogonal information. We synthesize these findings into a new set of activation domain predictions on human transcription factors.

Molecular dissection studies of TAC1, a transcription activator of Candida drug resistance genes of the human pathogenic fungus Candida albicans.

The up-regulation of ABC transporters Cdr1p and Cdr2p that efflux antifungal azole drugs are a leading cause of Multi-Drug Resistance (MDR) in the white fungus Candida albicans. C. albicans was reported to infect patients following the recent Covid-19 pandemic after they were given steroids for recovery. Previously, the TAC1 gene was identified as the transcriptional activator of Candida drug resistance genes (CDR1 and CDR2) and has no known human homologs. This makes it a good target for the development of novel antifungals. We, therefore, carried out the molecular dissection study of TAC1 to understand the functional regulation of the ABC transporter genes (CDR1 and CDR2) under its control. The N-terminal DNA Binding Domain (DBD) of Tac1p interacts with the Drug Responsive Element (DRE) present in the upstream promoter region of CDR1 and CDR2 genes of C. albicans. The interaction between DBD and DRE recruits Tac1p to the promoter of CDR genes. The C-terminal Acidic Activation Domain (AAD) of Tac1p interacts with the TATA box Binding Protein (TBP) and thus recruits TBP to the TATA box of CDR1 and CDR2 genes. Taking a cue from a previous study involving a TAC1 deletion strain that suggested that Tac1p acts as a xenobiotic receptor, in this study, we identified that the Middle Homology Region (MHR) of Tac1p acts as a probable xenobiotic binding domain (XBD) which plays an important role in Candida drug resistance. In addition, we studied the role of Tac1p in the regulation of some lipid profiling genes and stress response genes since they also contain the DRE consensus sequence and found that some of them can respond to xenobiotic stimuli.

Dynamic Solution Structures of Whole Human NAP1 Dimer Bound to One and Two Histone H2A-H2B Heterodimers Obtained by Integrative Methods

Nucleosome assembly protein 1 (NAP1) binds to histone H2A-H2B heterodimers, mediating their deposition on and eviction from the nucleosome. Human NAP1 (hNAP1) consists of a dimerization core domain and intrinsically disordered C-terminal acidic domain (CTAD), both of which are essential for H2A-H2B binding. Several structures of NAP1 proteins bound to H2A-H2B exhibit binding polymorphisms of the core domain, but the distinct structural roles of the core and CTAD domains remain elusive. Here, we have examined dynamic structures of the full-length hNAP1 dimer bound to one and two H2A-H2B heterodimers by integrative methods. Nuclear magnetic resonance (NMR) spectroscopy of full-length hNAP1 showed CTAD binding to H2A-H2B. Atomic force microscopy revealed that hNAP1 forms oligomers of tandem repeated dimers; therefore, we generated a stable dimeric hNAP1 mutant exhibiting the same H2A-H2B binding affinity as wild-type hNAP1. Size exclusion chromatography (SEC), multi-angle light scattering (MALS) and small angle X-ray scattering (SAXS), followed by modelling and molecular dynamics simulations, have been used to reveal the stepwise dynamic complex structures of hNAP1 binding to one and two H2A-H2B heterodimers. The first H2A-H2B dimer binds mainly to the core domain of hNAP1, while the second H2A-H2B binds dynamically to both CTADs. Based on our findings, we present a model of the eviction of H2A-H2B from nucleosomes by NAP1.

Tau protein binds to the P53 E3 ubiquitin ligase MDM2

Tau gene mutations cause a progressive dementia and neurotoxic Tau forms deposited in neurofibrillary tangles are hallmarks of neurodegenerative tauopathies. Loss of non-canonical Tau functions may contribute to disease. In fact, Tau depletion affects the cellular response to DNA damage and tauopathies exhibit the accumulation of DNA lesions. Moreover, Tau modulates P53 activity and cell fate. Considering that MDM2 is the main antagonist of P53, we investigated, using orthogonal assays, if Tau interacts with MDM2. We report the existence in cells and brain of a Tau-MDM2 complex that, in vitro, exhibits reduced P53 ubiquitination activity in a manner sensitive to a Tau mutation. The Tau-MDM2 interaction involves the microtubule-binding domain of Tau and the acidic domain of MDM2, reminiscent of the binding of Tau to negatively charged microtubules. Notably, MDM2 accumulates aberrantly in neurofibrillary tangles. Aging-associated insults may expose a novel loss-of-function of Tau in neurodegeneration and cancer.