Energy-driven genome regulation by ATP-dependent chromatin remodellers.

The packaging of DNA into chromatin in eukaryotes regulates gene transcription, DNA replication and DNA repair. ATP-dependent chromatin remodelling enzymes (re)arrange nucleosomes at the first level of chromatin organization. Their Snf2-type motor ATPases alter histone-DNA interactions through a common DNA translocation mechanism. Whether remodeller activities mainly catalyse nucleosome dynamics or accurately co-determine nucleosome organization remained unclear. In this Review, we discuss the emerging mechanisms of chromatin remodelling: dynamic remodeller architectures and their interactions, the inner workings of the ATPase cycle, allosteric regulation and pathological dysregulation. Recent mechanistic insights argue for a decisive role of remodellers in the energy-driven self-organization of chromatin, which enables both stability and plasticity of genome regulation - for example, during development and stress. Different remodellers, such as members of the SWI/SNF, ISWI, CHD and INO80 families, process (epi)genetic information through specific mechanisms into distinct functional outputs. Combinatorial assembly of remodellers and their interplay with histone modifications, histone variants, DNA sequence or DNA-bound transcription factors regulate nucleosome mobilization or eviction or histone exchange. Such input-output relationships determine specific nucleosome positions and compositions with distinct DNA accessibilities and mediate differential genome regulation. Finally, remodeller genes are often mutated in diseases characterized by genome dysregulation, notably in cancer, and we discuss their physiological relevance.

Histone acetylation and deacetylation - Mechanistic insights from structural biology.

Histones are subject to a diverse array of post-translational modifications. Among them, lysine acetylation is not only the most pervasive and dynamic modification but also highly consequential for regulating gene transcription. Although enzymes responsible for the addition and removal of acetyl groups were discovered almost 30 years ago, high-resolution structures of the enzymes in the context of their native complexes are only now beginning to become available, thanks to revolutionary technologies in protein structure determination and prediction. Here, we will review our current understanding of the molecular mechanisms of acetylation and deacetylation engendered by chromatin-modifying complexes, compare and contrast shared features, and discuss some of the pressing questions for future studies.

Cryo-EM structure of the Saccharomyces cerevisiae Rpd3L histone deacetylase complex.

The Rpd3L histone deacetylase (HDAC) complex is an ancient 12-subunit complex conserved in a broad range of eukaryotes that performs localized deacetylation at or near sites of recruitment by DNA-bound factors. Here we describe the cryo-EM structure of this prototypical HDAC complex that is characterized by as many as seven subunits performing scaffolding roles for the tight integration of the only catalytic subunit, Rpd3. The principal scaffolding protein, Sin3, along with Rpd3 and the histone chaperone, Ume1, are present in two copies, with each copy organized into separate lobes of an asymmetric dimeric molecular assembly. The active site of one Rpd3 is completely occluded by a leucine side chain of Rxt2, while the tips of the two lobes and the more peripherally associated subunits exhibit varying levels of flexibility and positional disorder. The structure reveals unexpected structural homology/analogy between unrelated subunits in the fungal and mammalian complexes and provides a foundation for deeper interrogations of structure, biology, and mechanism of these complexes, as well as for the discovery of HDAC complex-specific inhibitors.

Principles and functions of condensate modifying drugs.

Biomolecular condensates are compartmentalized communities of biomolecules, which unlike traditional organelles, are not enclosed by membranes. Condensates play roles in diverse cellular processes, are dysfunctional in many disease states, and are often enriched in classically "undruggable" targets. In this review, we provide an overview for how drugs can modulate condensate structure and function by phenotypically classifying them as dissolvers (dissolve condensates), inducers (induce condensates), localizers (alter localization of the specific condensate community members) or morphers (alter the physiochemical properties). We discuss the growing list of bioactive molecules that function as condensate modifiers (c-mods), including small molecules, oligonucleotides, and peptides. We propose that understanding mechanisms of condensate perturbation of known c-mods will accelerate the discovery of a new class of therapies for difficult-to-treat diseases.

Structure and flexibility of the yeast NuA4 histone acetyltransferase complex.

The NuA4 protein complex acetylates histones H4 and H2A to activate both transcription and DNA repair. We report the 3.1-Å resolution cryo-electron microscopy structure of the central hub of NuA4, which flexibly tethers the histone acetyltransferase (HAT) and Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) modules. The hub contains the large Tra1 subunit and a core that includes Swc4, Arp4, Act1, Eaf1, and the C-terminal region of Epl1. Eaf1 stands out as the primary scaffolding factor that interacts with the Tra1, Swc4, and Epl1 subunits and contributes the conserved HSA helix to the Arp module. Using nucleosome-binding assays, we find that the HAT module, which is anchored to the core through Epl1, recognizes H3K4me3 nucleosomes with hyperacetylated H3 tails, while the TINTIN module, anchored to the core via Eaf1, recognizes nucleosomes that have hyperacetylated H2A and H4 tails. Together with the known interaction of Tra1 with site-specific transcription factors, our data suggest a model in which Tra1 recruits NuA4 to specific genomic sites then allowing the flexible HAT and TINTIN modules to select nearby nucleosomes for acetylation.

Process Development for the Instant Quantification of Lycopene from Agricultural Produces Using Supercritical Fluid Chromatography-Diode Array Detector (SFC-DAD).

A quick, simple, and reliable isocratic ultra-performance supercritical fluid chromatography-photodiode array detector (UPSFC-DAD) method was developed and validated to determine lycopene in different horticultural products. The effects of stationary phase, co-solvent, pressure, temperature, flow rate, and mobile phase additive on the separation of lycopene were evaluated. The developed method involved BEH-2EP-2.1 × 150 mm, 5 µm as the stationary phase, and CO2/MeOH 85:15 (v/v) with formic acid as the additive at 0.10% as the mobile phase. The column temperature was maintained at 45 °C, ABPR at 1800 psi, and the mobile phase's flow rate was maintained at 1 mL/min. Under the optimized conditions, lycopene was successfully separated within 0.722 ± 0.001 min. The standard curve assayed over a range of 10 to 100 µg/mL resulted in a correlation coefficient of 0.998. The mean recoveries between 97.38% and 102.67% at different spiking levels with RSD < 2.5% were achieved. The intra and inter-day precision expressed as relative standard deviations (RSD) were found to range from 1.27% to 3.28% and from 1.57% to 4.18%, respectively. Robustness in terms of retention time (tR) and RSD were found to be 0.93 ± 0.23 min and less <2.80%, respectively. The limits of detection and quantification were 0.14 µg/mL and 0.37 µg/mL, respectively. This method was successfully applied to determine lycopene extracted from papaya, grapefruit, and bitter melon.

Process optimization for the supercritical carbondioxide extraction of lycopene from ripe grapefruit (Citrus paradisi) endocarp.

In this study, an underutilized citrus family fruit named grapefruit was explored for the extraction of lycopene using supercritical carbon dioxide (CO2) extraction technique. An experimental design was developed using response surface methodology to investigate the effect of supercritical carbon dioxide (CO2) operating parameter viz., pressure, temperature, CO2 flow rate, and extraction time on the extraction yield of lycopene yield from grapefruit. A total of 30 sets of experiments were conducted with six central points. The statistical model indicated that extraction pressure and extraction time individually, and their interaction, significantly affected the lycopene yield. The central composite design showed that the polynomial regression models developed were in agreement with the experimental results, with R2 of 0.9885. The optimum conditions for extraction of lycopene from grapefruit were 305 bar pressure, 35 g/min CO2 flow rate, 135 min of extraction time, and 70 °C temperature.

Phosphofructokinase relocalizes into subcellular compartments with liquid-like properties in vivo.

Although much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. We systematically examine the dynamic subcellular localization of glycolytic protein phosphofructokinase-1/PFK-1.1 in Caenorhabditis elegans. We determine that endogenous PFK-1.1 localizes to subcellular compartments in vivo. In neurons, PFK-1.1 forms phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in cytosolic dispersion of PFK-1.1. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Heterologous self-association domain cryptochrome 2 promotes formation of PFK-1.1 condensates and recruitment of aldolase/ALDO-1. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically form condensates in vivo.

Improved stability of phycobiliprotein within liposome stabilized by polyethylene glycol adsorbed cellulose nanocrystals.

This study ascertained the stability of phycobiliprotein (PBP), a bioactive protein from Dulse (Palmaria palmata) loaded within liposomes and stabilized with polyethylene glycol (2000 and 4000 g/mol) and desulfated CNCs (DCs) containing adsorbed polyethylene glycol (DCs-2000 and DCs-4000). The effect of pH, temperature and illumination on the stability of PBP was investigated. Results showed that the temperature had the most significant (p < 0.05) effect on the fluorescence intensity of the PBP, accounting for up to 70% loss of the fluorescence intensity for PBP loaded liposome (PL), PL stabilized with PEG-2000 (PLP-2000) and PEG 4000 (PLP-4000) and PL stabilized with desulfated CNCs (DCs), however, 60% for the PL stabilized with PEG 2000 and PEG 4000 adsorbed CNCs (PLDCs-2000 and PLDCs-4000) at 60 °C compared to those stabilized at 4 °C. A further increase in temperature to 80 °C led to a complete loss of fluorescence. Operating at the extreme pH's of 1.0 and 11.0 resulted in a loss of 90% and 30% fluorescence intensity, respectively for PBP in solution, whereas, 20% and 2% loss was observed for PBP incorporated inside the liposomes. Regarding the effect of illumination, PLDCs-2000 and PLDCs-4000 were the most stable, retaining the fluorescence intensity of PBP up to 70% after 72 h of exposure. This is compared to 85% loss of fluorescence for PBP in solution. Furthermore, at pH of 1.0, there was an increase in average particle size for the PLDCs-2000 and PLDCs-4000 from 189 ± 3 & 206 ± 2 nm to 6464 ± 211 & 6698 ± 317 nm and a charge reversal in the zeta potential from -36 ± 1 & -34 ± 2 to +16 ± 3 & +14 ± 1. Confocal and optical microscopic images confirmed the coalescence of PBP loaded liposome and agglomeration PLDCs-2000 and PLDCs-4000 under acidic pH (<3.0). In contrast, changes in temperature from 4 °C to 100 °C and illumination as a function of time up to 72 h resulted in no change in liposome size and zeta potential.

Tuning shape and internal structure of protein droplets via biopolymer filaments.

Macromolecules can phase separate to form liquid condensates, which are emerging as critical compartments in fields as diverse as intracellular organization and soft materials design. A myriad of macromolecules, including the protein FUS, form condensates which behave as isotropic liquids. Here, we investigate the influence of filament dopants on the material properties of protein liquids. We find that the short, biopolymer filaments of actin spontaneously partition into FUS droplets to form composite liquid droplets. As the concentration of the filament dopants increases, the coalescence time decreases, indicating that the dopants control viscosity relative to surface tension. The droplet shape is tunable and ranges from spherical to tactoid as the filament length or concentration is increased. We find that the tactoids are well described by a model of a quasi bipolar liquid crystal droplet, where nematic order from the anisotropic actin filaments competes with isotropic interfacial energy from the FUS, controlling droplet shape in a size-dependent manner. Our results demonstrate a versatile approach to construct tunable, anisotropic macromolecular liquids.

Development of microencapsulated anthocyanin-rich powder using soy protein isolate, jackfruit seed starch and an emulsifier (NBRE-15) as encapsulating materials.

A trend of present encapsulation research indicates an increased interest in the search for natural encapsulants for bioactive phytochemicals. The present study in pursuit of the same studies the use of jackfruit seed starch (JSS), an underutilized natural polysaccharide in conjugation with soy protein isolate (SPI) as an encapsulating material and NBRE-15 as an emulsifier. Three independent variables viz., total soluble solids (TSS, 20, 25 and 30° Brix), SPI: JSS (1:1, 1:3 and 1:5) and NBRE-15 (0.1, 0.2 and 0.3%) were optimized for achieving the most efficient encapsulation of anthocyanin using a three level, three parameter, Box-Behnken design (BBD) of the Design of Experiments (DOE). The responses considered for the optimization were monomeric anthocyanin content, antioxidant activity and encapsulation efficiency. A combination of 27.0% TSS, 1:5 SPI: JSS ratio and 0.3% NBRE-15 was found to be optimum for the encapsulation of anthocyanin with the desirability of 92.6%. Microcapsules obtained using the optimized combination of independent variables was found to contain 3215.59 mg/100 g monomeric anthocyanin. The antioxidant activity and encapsulation efficiency of the encapsulated material obtained using optimized combinations of independent variable were found to be 365.26 µmol Trolox/g and 89.71%, respectively. The microcapsules were also additionally analyzed for the particle size distribution and morphological characterization. Particle size analysis indicated that the microcapsules obtained had a mean particle size of 60.97 µm. Scanning electron microscopy for morphological characterization indicated that the microcapsules so obtained were oval to round in shape and had a smooth surface. Storage studies to estimate the half-life of anthocyanin in the microcapsule at room temperature (37 °C) clearly indicated greater stability i.e. 63 days when stored under amber-colored vial compared to only 35 days when stored under clear transparent vial.

Recent insights into the structure of TFIID, its assembly, and its binding to core promoter.

TFIID is a large multiprotein assembly that serves as a general transcription factor for transcription initiation by eukaryotic RNA polymerase II (Pol II). TFIID is involved in the recognition of the core promoter sequences and neighboring chromatin marks, and can interact with gene-specific activators and repressors. In order to obtain a better molecular and mechanistic understanding of the function of TFIID, its structure has been pursued for many years. However, the scarcity of TFIID and its highly flexible nature have made this pursuit very challenging. Recent breakthroughs, largely due to methodological advances in cryo-electron microscopy, have finally described the structure of this complex, both alone and engaged with core promoter DNA, revealing the functional significance of its conformational complexity in the process of core promoter recognition and initiation of Pol II transcription. Here, we review these recent structural insights and discuss their implications for our understanding of eukaryotic transcription initiation.

Architecture of the chromatin remodeler RSC and insights into its nucleosome engagement.

Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility. The Saccharomyces cerevisiae RSC (Remodeling the Structure of Chromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair. Here, we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored to this core by the N-terminus of Sth1. Our cryo-EM analysis of RSC bound to a nucleosome core particle shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of the Rsc8 SWIRM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes.

Supercritical fluid extraction of ß-carotene from ripe bitter melon pericarp.

Study ascertained the recovery of ß-carotene from enzyme-treated (enzyme load of 167 U/g) pericarp of ripe bitter melon using supercritical fluid extraction (SFE) technique. Effect of different pressure (ranged from 150-450 bar), carbon dioxide (CO2) flow rates (ranged from 15 to 55 ml/min), temperatures (from 50 to 90 °C), and extraction periods (from 45-225 minutes) were observed on the extraction efficiency of ß-carotene. Results showed that extraction pressure (X1) among extraction parameters had the most significant (p < 0.05) effect on extraction efficiency of the ß-carotene followed by allowed extraction time (X4), CO2 flow rate (X2) and the temperature of the extraction (X3). The maximum yield of 90.12% of ß-carotene from lyophilized enzymatic pretreated ripe bitter melon pericarp was achieved at the pressure of approx. 390 bar, flow rate of 35 mL/min, temperature at 70 °C and extraction time of 190 min, respectively. Based on the accelerated storage study the 70% retention shelf life of the ß-carotene into extract was estimated up to 2.27 months at 10 °C and up to 3.21 months at 5 °C.

Effect of different processing conditions on proximate composition, anti-oxidants, anti-nutrients and amino acid profile of grain sorghum.

The effect of different processing conditions (B: boiling; F: LAB fermentation; FS: fermentation and steaming; FSF: fermentation, steaming, flaking) of whole grain sorghum on the proximate composition, antioxidants, anti-nutrients, and amino acids (AAs) was evaluated. A marginal increase in the protein content and a decrease in the fat content was observed in the F-sample. Total phenolics reduced by 28%; DPPH scavenging activity and CUPRAC activity increased by 1.4 and 6 times, respectively during fermentation. Tannin content reduced by 30-39%, for the F, FS and FSF samples; highest reduction in trypsin inhibitory activity (58%) was observed in the FS-sample. Total AAs increased by 2.9 folds in FSF samples. Grain sorghum contained mostly hydrophobic AAs (30-34%). The ratio of Essential amino acid to total amino acid and predicted protein efficiency ratio were highest in the F-sample, whereas predicted biological value of the FSF was 3 times than that of the control.

Structure of human TFIID and mechanism of TBP loading onto promoter DNA.

The general transcription factor IID (TFIID) is a critical component of the eukaryotic transcription preinitiation complex (PIC) and is responsible for recognizing the core promoter DNA and initiating PIC assembly. We used cryo-electron microscopy, chemical cross-linking mass spectrometry, and biochemical reconstitution to determine the complete molecular architecture of TFIID and define the conformational landscape of TFIID in the process of TATA box-binding protein (TBP) loading onto promoter DNA. Our structural analysis revealed five structural states of TFIID in the presence of TFIIA and promoter DNA, showing that the initial binding of TFIID to the downstream promoter positions the upstream DNA and facilitates scanning of TBP for a TATA box and the subsequent engagement of the promoter. Our findings provide a mechanistic model for the specific loading of TBP by TFIID onto the promoter.

Structural and Functional Studies of the Daunorubicin Priming Ketosynthase DpsC.

Daunorubicin is a type II polyketide, one of a large class of polyaromatic natural products with anticancer, antibiotic, and antiviral activity. Type II polyketides are formed by the assembly of malonyl-CoA building blocks, though in rare cases, biosynthesis is initiated by the incorporation of a nonmalonyl derived starter unit, which adds molecular diversity to the poly-ß-ketone backbone. Priming mechanisms for the transfer of novel starter units onto polyketide synthases (PKS) are still poorly understood. Daunorubicin biosynthesis incorporates a unique propionyl starter unit thought to be selected for by a subclass ("DpsC type") of priming ketosynthases (KS III). To date, however, no structural information exists for this subclass of KS III enzymes. Although selectivity for self-acylation with propionyl-CoA has previously been implied, we demonstrate that DpsC shows no discrimination for self-acylation or acyl-transfer to the cognate acyl carrier protein, DpsG with short acyl-CoAs. We present five crystal structures of DpsC, including apo-DpsC, acetyl-DpsC, propionyl-DpsC, butyryl-DpsC, and a cocrystal of DpsC with a nonhydrolyzable phosphopantetheine (PPant) analogue. The DpsC crystal structures reveal the architecture of the active site, the molecular determinants for catalytic activity and homology to O-malonyl transferases, but also indicate distinct differences. These results provide a structural basis for rational engineering of starter unit selection in type II polyketide synthases.

Towards a mechanistic understanding of core promoter recognition from cryo-EM studies of human TFIID.

TFIID is a critical component of the eukaryotic transcription pre-initiation complex (PIC) required for the recruitment of RNA Pol II to the start site of protein-coding genes. Within the PIC, TFIID's role is to recognize and bind core promoter sequences and recruit the rest of the PIC components. Due to its size and its conformational complexity, TFIID poses a serious challenge for structural characterization. The small amounts of purified TFIID that can be obtained by present methods of purification from endogenous sources has limited structural studies to cryo-EM visualization, which requires very small amounts of sample. Previous cryo-EM studies have shed light on how the extreme conformational flexibility of TFIID is involved in core promoter DNA binding. Recent progress in cryo-EM methodology has facilitated a parallel progress in the study of human TFIID, leading to an improvement in resolution and the identification of the structural elements in the complex directly involved in DNA interaction. While many questions remain unanswered, the present structural knowledge of human TFIID suggests a mechanism for the sequential engagement with different core promoter sequences and how it could be influenced by regulatory factors.

ATP as a biological hydrotrope.

Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions. Typically, hydrotropes are amphiphilic molecules and differ from classical surfactants in that they have low cooperativity of aggregation and work at molar concentrations. Here, we show that adenosine triphosphate (ATP) has properties of a biological hydrotrope. It can both prevent the formation of and dissolve previously formed protein aggregates. This chemical property is manifested at physiological concentrations between 5 and 10 millimolar. Therefore, in addition to being an energy source for biological reactions, for which micromolar concentrations are sufficient, we propose that millimolar concentrations of ATP may act to keep proteins soluble. This may in part explain why ATP is maintained in such high concentrations in cells.

An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function.

Stress granules (SG) are membrane-less compartments involved in regulating mRNAs during stress. Aberrant forms of SGs have been implicated in age-related diseases, such as amyotrophic lateral sclerosis (ALS), but the molecular events triggering their formation are still unknown. Here, we find that misfolded proteins, such as ALS-linked variants of SOD1, specifically accumulate and aggregate within SGs in human cells. This decreases the dynamics of SGs, changes SG composition, and triggers an aberrant liquid-to-solid transition of in vitro reconstituted compartments. We show that chaperone recruitment prevents the formation of aberrant SGs and promotes SG disassembly when the stress subsides. Moreover, we identify a backup system for SG clearance, which involves transport of aberrant SGs to the aggresome and their degradation by autophagy. Thus, cells employ a system of SG quality control to prevent accumulation of misfolded proteins and maintain the dynamic state of SGs, which may have relevance for ALS and related diseases.