Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation.

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

Delving into human α1,4-galactosyltransferase acceptor specificity: The role of enzyme dimerization.

Human α1,4-galactosyltransferase (A4galt), a Golgi apparatus-resident GT, synthesizes Gb3 glycosphingolipid (GSL) and P1 glycotope on glycoproteins (GPs), which are receptors for Shiga toxin types 1 and 2. Despite the significant role of A4galt in glycosylation processes, the molecular mechanisms underlying its varied acceptor specificities remain poorly understood. Here, we attempted to elucidate A4galt specificity towards GSLs and GPs by exploring its interaction with GTs with various acceptor specificities, GP-specific ß1,4-galactosyltransferase 1 (B4galt1) and GSL-specific ß1,4-galactosyltransferase isoenzymes 5 and 6 (B4galt5 and B4galt6). Using a novel NanoBiT assay, we found that A4galt can form homodimers and heterodimers with B4galt1 and B4galt5 in two cell lines, human embryonic kidney cells (HEK293T) and Chinese hamster ovary cells (CHO-Lec2). We found that A4galt-B4galts heterodimers preferred N-terminally tagged interactions, while in A4galt homodimers, the favored localization of the fused tag depended on the cell line used. Furthermore, by employing AlphaFold for state-of-the-art structural prediction, we analyzed the interactions and structures of these enzyme complexes. Our analysis highlighted that the A4galt-B4galt5 heterodimer exhibited the highest prediction confidence, indicating a significant role of A4galt heterodimerization in determining enzyme specificity toward GSLs and GPs. These findings enhance our knowledge of A4galt acceptor specificity and may contribute to a better comprehension of pathomechanisms of the Shiga toxin-related diseases.

Enzyme Is the Name-Adapter Is the Game.

Signaling proteins in eukaryotes usually comprise a catalytic domain coupled to one or several interaction domains, such as SH2 and SH3 domains. An additional class of proteins critically involved in cellular communication are adapter or scaffold proteins, which fulfill their purely non-enzymatic functions by organizing protein-protein interactions. Intriguingly, certain signaling enzymes, e.g., kinases and phosphatases, have been demonstrated to promote particular cellular functions by means of their interaction domains only. In this review, we will refer to such a function as "the adapter function of an enzyme". Though many stories can be told, we will concentrate on several proteins executing critical adapter functions in cells of the immune system, such as Bruton´s tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K), and SH2-containing inositol phosphatase 1 (SHIP1), as well as in cancer cells, such as proteins of the rat sarcoma/extracellular signal-regulated kinase (RAS/ERK) mitogen-activated protein kinase (MAPK) pathway. We will also discuss how these adaptor functions of enzymes determine or even undermine the efficacy of targeted therapy compounds, such as ATP-competitive kinase inhibitors. Thereby, we are highlighting the need to develop pharmacological approaches, such as proteolysis-targeting chimeras (PROTACs), that eliminate the entire protein, and thus both enzymatic and adapter functions of the signaling protein. We also review how genetic knock-out and knock-in approaches can be leveraged to identify adaptor functions of signaling proteins.

Synthetic Dimerization Approaches for In Vivo Studies in Saccharomyces cerevisiae.

Synthetic tethering approaches induced by chemical means offer precise control over protein interactions in cells. They enable the manipulation of when, where, and how proteins interact, making it possible to study their functions, dynamics, and cellular consequences at a molecular level. These methods are versatile, reversible, and adaptable, allowing the dissection of complex cellular processes and the engineering of cellular functions. Here, we describe two chemically induced dimerization systems in the model organism Saccharomyces cerevisiae. Using the autophagy pathway as an example, we show how these approaches can be used to dissect molecular events in cells.

Studying Selective Autophagy of Protein Aggregates Using Particles Induced by Multimerization (PIMs).

Selective autophagy of protein aggregates, called aggrephagy, is vital for maintaining cellular homeostasis. Classically, studying aggrephagy has been challenging due to the infrequent occurrence of autophagic events and the lack of control over the specificity and timing of protein aggregation. We previously reported two variants of a PIM (particles induced by multimerization) assay that enable the formation of chemically induced, fluorescently labeled protein aggregates in cells. PIMs are recognized by the selective autophagy machinery and are subsequently degraded in the lysosome. By making use of pH-sensitive fluorescent proteins, such as GFP or mKeima, the PIM assay allows for direct visualization of aggregate clearance in cells. Here, we describe a protocol for the use of the PIM assay to study aggrephagy in live and fixed cells.

Analysis of homodimer formation in 12-oxophytodienoate reductase 3 in solutio and crystallo challenges the physiological role of the dimer.

12-oxophytodienoate reductase 3 (OPR3) is a key enzyme in the biosynthesis of jasmonoyl-L-isoleucine, the receptor-active form of jasmonic acid and crucial signaling molecule in plant defense. OPR3 was initially crystallized as a self-inhibitory dimer, implying that homodimerization regulates enzymatic activity in response to biotic and abiotic stresses. Since a sulfate ion is bound to Y364, mimicking a phosphorylated tyrosine, it was suggested that dimer formation might be controlled by reversible phosphorylation of Y364 in vivo. To investigate OPR3 homodimerization and its potential physiological role in more detail, we performed analytical gel filtration and dynamic light scattering on wild-type OPR3 and three variants (R283D, R283E, and Y364P). The experiments revealed a rapid and highly sensitive monomer-dimer equilibrium for all OPR3 constructs. We crystallized all constructs with and without sulfate to examine its effect on the dimerization process and whether reversible phosphorylation of Y364 triggers homodimerization in vivo. All OPR3 constructs crystallized in their monomeric and dimeric forms independent of the presence of sulfate. Even variant Y364P, lacking the putative phosphorylation site, was crystallized as a self-inhibitory homodimer, indicating that Y364 is not required for dimerization. Generally, the homodimer is relatively weak, and our results raise doubts about its physiological role in regulating jasmonate biosynthesis.

Fine-Tuning the Epigenetic Landscape: Chemical Modulation of Epigenome Editors.

Epigenome editing has emerged as a powerful technique for targeted manipulation of the chromatin and transcriptional landscape, employing designer DNA binding domains fused with effector domains, known as epi-editors. However, the constitutive expression of dCas9-based epi-editors presents challenges, including off-target activity and lack of temporal resolution. Recent advancements of dCas9-based epi-editors have addressed these limitations by introducing innovative switch systems that enable temporal control of their activity. These systems allow precise modulation of gene expression over time and offer a means to deactivate epi-editors, thereby reducing off-target effects associated with prolonged expression. The development of novel dCas9 effectors regulated by exogenous chemical signals has revolutionized temporal control in epigenome editing, significantly expanding the researcher's toolbox. Here, we provide a comprehensive review of the current state of these cutting-edge systems and specifically discuss their advantages and limitations, offering context to better understand their capabilities.

An overview of RAF kinases and their inhibitors (2019-2023).

Protein kinases (PKs) including RAF, perform a principal role in regulating countless cellular events such as cell growth, differentiation, and angiogenesis. Overexpression and mutation of RAF kinases are significant contributors to the development and spread of cancer. Therefore, RAF kinase inhibitors show promising outcomes as anti-cancer small molecules by suppressing the expression of RAF protein, blocking RAS/RAF interaction, or inhibiting RAF enzymes. Currently, there are insufficient reports about approving drugs with minimal degree of toxicity. Therefore, it is an urgent need to develop new RAF kinase inhibitors correlated with increased anticancer activity and lower cytotoxicity. This review outlines reported RAF kinase inhibitors for cancer treatment in patents and literature from 2019 to 2023. It highlights the available inhibitors by shedding light on their chemical structures, biochemical profiles, and current status. Additionally, we highlighted the hinge region-binding moiety of the reported compounds by showing the hydrogen bond patterns of representative inhibitors with the hinge region for each class. In recent years, RAF kinase inhibitors have gained considerable attention in cancer research and drug development due to their potential to be studied under clinical trials and their demonstration of various degrees of efficacy and safety profiles across different cancer types. However, addressing challenges related to drug resistance and safety represents a major avenue for the optimization and enhancement of RAF kinase inhibitors. Strategies to overcome such obstacles were discussed such as developing novel pan-RAF inhibitors, RAF dimer inhibitors, and combination treatments.

Ab initio investigation of the geometrical behavior in solution and electronic structure of the anion complexes [bis(1,3-dithiole-2-thione-4,5-dithiolate)M], for M = Bi(III), Sb(III), and Zn(II).

CONTEXT: 1,3-Dithiole-2-thione-4,5-dithiolate (dmit) ligands are known for their conductive and optical properties. Dmit compounds have been assessed for use in sensor devices, information storage, spintronics, and optical material applications. Associations with various metallic centers endow dmit complexes with magnetic, optical, conductive, and antioxidant properties. Optical doping can facilitate the fabrication of magnetic conductor materials from ground-state nonmagnetic cations. While most studied complexes involve transition-metal centers due to their diverse chemistry, compounds with representative elements are less explored in the literature. This study investigated the structural and electronic properties of bisdmit complexes with representative Bi(III), Sb(III), and Zn(II) cations. AIMD calculations revealed two new geometries for Bi(III) and Zn(II) complexes, diverging from the isolated geometry typically used in quantum chemical calculations. The coordination of acetonitrile molecules to the cationic centers of the complexes resulted in unstable structures, while the dimerization of the complexes was stable. SA-CASSCF/NEVPT2 calculations were applied to the structures of the isolated complexes and stable dimers, confirming the multireference character of the electronic structure of the three systems and the multiconfigurational character of the Bi(III) complex. The electronic spectra simulated by the STEOM-DLPNO-CCSD calculations accurately reproduced the experimental UV‒Vis spectra indicating the participation of the isolated Bi(III) dmit complex and its dimeric form in solution. METHODOLOGY: AIMD calculations of the dmit salts were conducted using the GFN2-xTB method with 60 explicit acetonitrile molecules as the solvent at 300 K for a total simulation time of 50.0 ps, with printing intervals of 0.5 fs. The final geometries were optimized employing the PBEh-3c compound method, incorporating implicit conductor-like polarizable continuum model (CPCM) solvation for acetonitrile. Local energy decomposition (LED) analysis at the DLPNO-CCSD(T)/Def2-TZVP level of theory was utilized to investigate the stability of the complex geometries identified by AIMD. The electronic structures of the complexes were assessed using the SA-CASSCF/NEVPT2/Def2-TZVP method to confirm the multiconfigurational and multireference nature of their electronic structures. Electronic spectra were analyzed using the STEOM-DLPNO-CCSD/Def2-TZVP method, with CPCM used to simulate an acetonitrile medium.

The N-terminal coiled-coil domain of Arabidopsis CROWDED NUCLEI 1 is required for nuclear morphology maintenance.

The Arabidopsis CROWDED NUCLEI (CRWN) family proteins form a lamina-like meshwork beneath the nuclear envelope with multiple functions, including maintenance of nuclear morphology, genome organization, DNA damage repair and transcriptional regulation. CRWNs can form homodimers/heterodimers through protein‒protein interactions; however, the exact molecular mechanism of CRWN dimer formation and the diverse functions of different CRWN domains are not clear. In this report, we show that the N-terminal coiled-coil domain of CRWN1 facilitates its homodimerization and heterodimerization with the coiled-coil domains of CRWN2-CRWN4. We further demonstrated that the N-terminus but not the C-terminus of CRWN1 is sufficient to rescue the defect in nuclear morphology of the crwn1 crwn2 mutant to the WT phenotype. Moreover, both the N- and C-terminal fragments of CRWN1 are necessary for its normal function in the regulation of plant development. Collectively, our data shed light on the mechanism of plant lamina network formation and the functions of different domains in plant lamin-like proteins.

Double Chalcogen Bonding Recognition Arrays in Solution.

N-substituted pyridino-based congeners of Ebselen, named here as Pyrselen, incorporating proximal Se and N atoms, undergo dimerization in solution and in the solid state through a dual donor-acceptor arrangement of chalcogen bonding sites. Dimerization constants were measured within the 15-50 M-1 range. Computational studies on the dimers depict a notable charge-transfer contribution to the association, validating Pyrselen as an effective scaffold for designing chalcogen-bonding-based recognition motifs. Insert abstract text here.

Dimerization of Full-length Aß -42 Peptide: A Comparison of Different Force Fields and Water Models.

Among the two isoforms of amyloid- i.e., Aß-40 and Aß-42, Aß-42 is more toxic due to its increased aggregation propensity. The oligomerization pathways of amyloid-ß may be investigated by studying its dimerization process at an atomic level. Intrinsically disordered proteins (IDPs) lack well-defined structures and are associated with numerous neurodegenerative disorders. Molecular dynamics simulations of these proteins are often limited by the choice of parameters due to inconsistencies in the empirically developed protein force fields and water models. To evaluate the accuracy of recently developed force fields for IDPs, we study the dimerization of full-length Aß-42 in aqueous solution with three different combinations of AMBER force field parameters and water models such as ff14SB/TIP3P, ff19SB/OPC, and ff19SB/TIP3P using classical MD and Umbrella Sampling method. This work may be used as a benchmark to compare the performance of different force fields for the simulations of IDPs.

Controlling the Subcellular Localization of Signaling Proteins Using Chemically Induced Dimerization and Optogenetics.

A given protein can perform numerous roles in a cell with its participation in protein complexes and distinct localization within the cell playing a critical role in its diverse functions. Thus, the ability to artificially dimerize proteins and recruit proteins to specific locations in a cell has become a powerful tool for the investigation of protein function and the understanding of cell biology. Here, we discuss two systems that have been used to activate signal transduction pathways, a chemically inducible dimerization (CID) and a light-inducible (LI) system to control signaling and cytoskeletal regulation in a spatial and temporal manner.

Regioselective Oxidative Phenol Coupling by a Mushroom Unspecific Peroxygenase.

Bioactive dimeric (pre-)anthraquinones are ubiquitous in nature. Their biosynthesis via an oxidative phenol coupling (OPC) step is catalyzed by either cytochrome P450 enzymes, peroxidases, or laccases. While the biocatalysis of OPC in molds (Ascomycota) is well-known, the respective enzymes of mushroom-forming fungi (Basidiomycota) are still unknown. Here, we report on the biosynthesis of the atropisomers phlegmacin A1 and B1, unsymmetrical 7,10'-homo-coupled dihydroanthracenones of the mushroom Cortinarius odorifer. The biosynthesis was heterologously reconstituted in the mold Aspergillus niger. We show that methylation of the dimeric (pre-)anthraquinone building block atrochrysone to its 6-O-methyl ether torosachrysone by the O-methyltransferase (CoOMT1) precedes the regioselective homo-coupling to phlegmacin, catalyzed by an unspecific peroxygenase (CoUPO1). Our results revealed an unprecedented UPO-mediated unsymmetric OPC reaction, thereby expanding the biocatalytic portfolio of OPC-type reactions beyond the commonly reported enzymes. The findings highlight the pivotal role of OPC in natural processes, demonstrating that Basidiomycota employed peroxygenases to develop the ability to selectively couple aryls, distinct and convergent to any other group of organisms.

Highly Efficient Low-loaded PdOx/AlSiOx for Ethylene Dimerization.

Ethylene dimerization is an industrial process that is currently carried out using homogeneous catalysts. Here we present a highly active heterogeneous catalyst containing minute amounts of atomically dispersed Pd. It requires no co-catalyst(s) or activator(s) and significantly outperforms previously reported catalysts tested under similar reaction conditions. The selectivity to C4- and C6-hydrocarbons was about 80% and 10% at 42% ethylene conversion at 200°C using an industrially relevant feed containing 50 vol% ethylene, respectively. Our kinetic and catalyst characterization experiments complemented by density functional theory calculations provide molecular insights into the local environment of isolated Pd(II)Ox species and their role in achieving high activity in the target reaction. When the developed catalyst was rationally integrated with a Mo-containing olefin metathesis catalyst in the same reactor, the formed butenes reacted with ethylene to propylene with a selectivity of 98% at about 24% ethylene conversion.

Structural Analysis of the Drosophila melanogaster GSTome.

Glutathione transferase (GST) is a superfamily of ubiquitous enzymes, multigenic in numerous organisms and which generally present homodimeric structures. GSTs are involved in numerous biological functions such as chemical detoxification as well as chemoperception in mammals and insects. GSTs catalyze the conjugation of their cofactor, reduced glutathione (GSH), to xenobiotic electrophilic centers. To achieve this catalytic function, GSTs are comprised of a ligand binding site and a GSH binding site per subunit, which is very specific and highly conserved; the hydrophobic substrate binding site enables the binding of diverse substrates. In this work, we focus our interest in a model organism, the fruit fly Drosophila melanogaster (D. mel), which comprises 42 GST sequences distributed in six classes and composing its GSTome. The goal of this study is to describe the complete structural GSTome of D. mel to determine how changes in the amino acid sequence modify the structural characteristics of GST, particularly in the GSH binding sites and in the dimerization interface. First, we predicted the 3D atomic structures of each GST using the AlphaFold (AF) program and compared them with X-ray crystallography structures, when they exist. We also characterized and compared their global and local folds. Second, we used multiple sequence alignment coupled with AF-predicted structures to characterize the relationship between the conservation of amino acids in the sequence and their structural features. Finally, we applied normal mode analysis to estimate thermal B-factors of all GST structures of D. mel. Particularly, we extracted flexibility profiles of GST and identify key residues and motifs that are systematically involved in the ligand binding/dimerization processes and thus playing a crucial role in the catalytic function. This methodology will be extended to guide the in silico design of synthetic GST with new/optimal catalytic properties for detoxification applications.

Structural and Thermodynamic Insights into Dimerization Interfaces of Drosophila Glutathione Transferases.

This study presents a comprehensive analysis of the dimerization interfaces of fly GSTs through sequence alignment. Our investigation revealed GSTE1 as a particularly intriguing target, providing valuable insights into the variations within Delta and Epsilon GST interfaces. The X-ray structure of GSTE1 was determined, unveiling remarkable thermal stability and a distinctive dimerization interface. Utilizing circular dichroism, we assessed the thermal stability of GSTE1 and other Drosophila GSTs with resolved X-ray structures. The subsequent examination of GST dimer stability correlated with the dimerization interface supported by findings from X-ray structural analysis and thermal stability measurements. Our discussion extends to the broader context of GST dimer interfaces, offering a generalized perspective on their stability. This research enhances our understanding of the structural and thermodynamic aspects of GST dimerization, contributing valuable insights to the field.

Structural Characterization of Disulfide-Linked p53-Derived Peptide Dimers.

Disulfide bonds provide a convenient method for chemoselective alteration of peptide and protein structure and function. We previously reported that mild oxidation of a p53-derived bisthiol peptide (CTFANLWRLLAQNC) under dilute non-denaturing conditions led to unexpected disulfide-linked dimers as the exclusive product. The dimers were antiparallel, significantly α-helical, resistant to protease degradation, and easily reduced back to the original bisthiol peptide. Here we examine the intrinsic factors influencing peptide dimerization using a combination of amino acid substitution, circular dichroism (CD) spectroscopy, and X-ray crystallography. CD analysis of peptide variants suggests critical roles for Leu6 and Leu10 in the formation of stable disulfide-linked dimers. The 1.0 Å resolution crystal structure of the peptide dimer supports these data, revealing a leucine-rich LxxLL dimer interface with canonical knobs-into-holes packing. Two levels of higher-order oligomerization are also observed in the crystal: an antiparallel "dimer of dimers" mediated by Phe3 and Trp7 residues in the asymmetric unit and a tetramer of dimers mediated by Trp7 and Leu10. In CD spectra of Trp-containing peptide variants, minima at 227 nm provide evidence for the dimer of dimers in dilute aqueous solution. Importantly, and in contrast to the original dimer model, the canonical leucine-rich core and robust dimerization of most peptide variants suggests a tunable molecular architecture to target various proteins and evaluate how folding and oligomerization impact various properties, such as cell permeability.

Probing Self-Assembly of Ammeline in Chloroform and Aqueous Media: Interplay Between Hydrogen Bonding Diversity and Dimerization.

Ammeline (AM) is a molecule with a very low reputation in the field of supramolecular community, but with a recently proven potential both experimentally and theoretically. In this work, dispersion-corrected density functional theory (DFT-D) computations and molecular dynamics (MD) simulations were employed to understand the aggregation mechanism of AM in chloroform and water media. Our DFT-D and MD analyzes show that the most important interactions are those formed by the amine groups (-NH2) with both the pyridine-type nitrogen atoms and the carbonyl groups (C=O). In the more polar solvent, the interactions between water molecules and the C=O group prevent the AM from forming more interactions with itself. Nevertheless, four types of dimers involving N-H∙∙∙O interactions were found to exist in water solutions. The overlooked tetrel bond between endocyclic N and C atoms can also stabilize dimers in solution. Moreover, while most AM dimers are enthalpy-driven, our results indicate that the unique DD-AA dimer (D=donor, A=acceptor) that originates cyclic rosettes is entropy-driven.

Zwitterionic nanoparticles from indocyanine green dimerization for imaging-guided cancer phototherapy.

Indocyanine green (ICG), the only near-infrared (NIR) dye approved for clinical use, has received increasing attention as a theranostic agent wherein diagnosis (fluorescence) is combined with therapy (phototherapy), but suffers rapid hepatic clearance, poor photostability, and limited accumulation at tumor sites. Here we report that dimerized ICG can self-assemble to form zwitterionic nanoparticles (ZN-dICG), which generate fluorescence self-quenching but exhibit superior photothermal and photodynamic properties over ICG. The zwitterionic moieties confer ZN-dICG an ultralow critical micelle concentration and high colloidal stability with low non-specific binding in vivo. In addition, ZN-dICG can respond to the over-generated reactive oxygen species (ROSs) and dissociate to restore NIR fluorescence of ICG, amplifying the sensitivity via albumin binding for low-background imaging of tumors. Following systemic administration, ZN-dICG accumulated in tumors of xenograft-bearing mice for imaging primary and metastatic tumors, and induced tumor ablation under laser irradiation. The discovery of ZN-dICG would contribute to the design of translational phototheranostic platform with high biocompatibility. STATEMENT OF SIGNIFICANCE: Indocyanine green (ICG) has been extensively studied as a phototheranostic agent that combines imaging with phototherapies, but it suffers from rapid hepatic clearance, poor photostability, and limited accumulation at tumor sites. Here, we report a strategy to construct ICG dimers (ICG-tk-ICG) by conjugating two ICG molecules via a thioketal bond, which can self-assemble into zwitterionic nanoparticles (ZN-dICG) at ultralow critical micelle concentrations, exhibiting superior photothermal and photodynamic properties over ICG. ZN-dICG responds to the over-generated ROS in tumors and dissociates to restore the NIR fluorescence of ICG, enhancing the sensitivity via albumin binding for low-background imaging of tumors. This study offers a supramolecular strategy that may potentiate the clinical translation of ICG in imaging-guided cancer phototherapy.