Atomistic description of the OCTN1 recognition mechanism via in silico methods.

The Organic Cation Transporter Novel 1 (OCTN1), also known as SLC22A4, is widely expressed in various human tissues, and involved in numerous physiological and pathological processes remains. It facilitates the transport of organic cations, zwitterions, with selectivity for positively charged solutes. Ergothioneine, an antioxidant compound, and acetylcholine (Ach) are among its substrates. Given the lack of experimentally solved structures of this protein, this study aimed at generating a reliable 3D model of OCTN1 to shed light on its substrate-binding preferences and the role of sodium in substrate recognition and transport. A chimeric model was built by grafting the large extracellular loop 1 (EL1) from an AlphaFold-generated model onto a homology model. Molecular dynamics simulations revealed domain-specific mobility, with EL1 exhibiting the highest impact on overall stability. Molecular docking simulations identified cytarabine and verapamil as highest affinity ligands, consistent with their known inhibitory effects on OCTN1. Furthermore, MM/GBSA analysis allowed the categorization of substrates into weak, good, and strong binders, with molecular weight strongly correlating with binding affinity to the recognition site. Key recognition residues, including Tyr211, Glu381, and Arg469, were identified through interaction analysis. Ach demonstrated a low interaction energy, supporting the hypothesis of its one-directional transport towards to outside of the membrane. Regarding the role of sodium, our model suggested the involvement of Glu381 in sodium binding. Molecular dynamics simulations of systems at increasing levels of Na+ concentrations revealed increased sodium occupancy around Glu381, supporting experimental data associating Na+ concentration to molecule transport. In conclusion, this study provides valuable insights into the 3D structure of OCTN1, its substrate-binding preferences, and the role of sodium in the recognition. These findings contribute to the understanding of OCTN1 involvement in various physiological and pathological processes and may have implications for drug development and disease management.

In Silico Description of the Direct Inhibition Mechanism of Endothelial Lipase by ANGPTL3.

Angiopoietin-like protein 3 (ANGPTL3) is a plasmatic protein that plays a crucial role in lipoprotein metabolism by inhibiting the lipoprotein lipase (LPL) and the endothelial lipase (EL) responsible for the hydrolysis of phospholipids on high-density lipoprotein (HDL). Interest in developing new pharmacological therapies aimed at inhibiting ANGPTL3 has been growing due to the hypolipidemic and antiatherogenic profile observed in its absence. The goal of this study was the in silico characterization of the interaction between ANGPTL3 and EL. Because of the lack of any structural information on both the trimeric coiled-coil N-terminal domain of ANGPTL3 and the EL homodimer as well as data regarding their interactions, the first step was to obtain the three-dimensional model of these two proteins. The models were then refined via molecular dynamics (MD) simulations and used to investigate the interaction mechanism. The analysis of interactions in different docking poses and their refinement via MD allowed the identification of three specific glutamates of ANGPTL3 that recognize a positively charged patch on the surface of EL. These ANGPTL3 key residues, i.e., Glu154, Glu157, and Glu160, could form a putative molecular recognition site for EL. This study paves the way for future investigations aimed at confirming the recognition site and at designing novel inhibitors of ANGPTL3.

Caulobacter segnis Dioxygenase CsO2: A Practical Biocatalyst for Stilbenoid Ozonolysis.

Ozonolysis is a useful as well as dangerous reaction for performing alkene cleavage. On the other hand, enzymes are considered a more sustainable and safer alternative. Among them, Caulobacter segnis dioxygenase (CsO2) known so far for its ability to catalyze the coenzyme-free oxidation of vinylguaiacol into vanillin, was selected and its substrate scope evaluated towards diverse natural and synthetic stilbenoids. Under optimized conditions, CsO2 catalyzed the oxidative cleavage of the C=C double bonds of various trans-stilbenes, providing that a hydroxyl moiety was necessary in para-position of the phenyl group (e. g., resveratrol and its derivatives) for the reaction to take place, which was confirmed by modelling studies. The reactions occurred rapidly (0.5-3 h) with high conversions (95-99 %) and without formation of by-products. The resveratrol biotransformation was carried out on 50-mL scale thus confirming the feasibility of the biocatalytic system as a preparative method.

Effect of Fc core fucosylation and light chain isotype on IgG1 flexibility.

N-glycosylation plays a key role in modulating the bioactivity of monoclonal antibodies (mAbs), as well as the light chain (LC) isotype can influence their physicochemical properties. However, investigating the impact of such features on mAbs conformational behavior is a big challenge, due to the very high flexibility of these biomolecules. In this work we investigate, by accelerated molecular dynamics (aMD), the conformational behavior of two commercial immunoglobulins G1 (IgG1), representative of κ and λ LCs antibodies, in both their fucosylated and afucosylated forms. Our results show, through the identification of a stable conformation, how the combination of fucosylation and LC isotype modulates the hinge behavior, the Fc conformation and the position of the glycan chains, all factors potentially affecting the binding to the FcγRs. This work also represents a technological enhancement in the conformational exploration of mAbs, making aMD a suitable approach to clarify experimental results.

HSPB8 frameshift mutant aggregates weaken chaperone-assisted selective autophagy in neuromyopathies.

Chaperone-assisted selective autophagy (CASA) is a highly selective pathway for the disposal of misfolding and aggregating proteins. In muscle, CASA assures muscle integrity by favoring the turnover of structural components damaged by mechanical strain. In neurons, CASA promotes the removal of aggregating substrates. A crucial player of CASA is HSPB8 (heat shock protein family B (small) member 8), which acts in a complex with HSPA, their cochaperone BAG3, and the E3 ubiquitin ligase STUB1. Recently, four novel HSPB8 frameshift (fs) gene mutations have been linked to neuromyopathies, and encode carboxy-terminally mutated HSPB8, sharing a common C-terminal extension. Here, we analyzed the biochemical and functional alterations associated with the HSPB8_fs mutant proteins. We demonstrated that HSPB8_fs mutants are highly insoluble and tend to form proteinaceous aggregates in the cytoplasm. Notably, all HSPB8 frameshift mutants retain their ability to interact with CASA members but sequester them into the HSPB8-positive aggregates together with two autophagy receptors SQSTM1/p62 and TAX1BP1. This copartitioning process negatively affects the CASA capability to remove its clients and causes a general failure in proteostasis response. Further analyses revealed that the aggregation of the HSPB8_fs mutants occurs independently of the other CASA members or from the autophagy receptors interaction, but it is an intrinsic feature of the mutated amino acid sequence. HSPB8_fs mutants aggregation alters the differentiation capacity of muscle cells and impairs sarcomere organization. Collectively, these results shed light on a potential pathogenic mechanism shared by the HSPB8_fs mutants described in neuromuscular diseases.Abbreviations : ACD: α-crystallin domain; ACTN: actinin alpha; BAG3: BAG cochaperone 3; C: carboxy; CASA: chaperone-assisted selective autophagy; CE: carboxy-terminal extension; CLEM: correlative light and electron microscopy; CMT2L: Charcot-Marie-Tooth type 2L; CTR: carboxy-terminal region; dHMNII: distal hereditary motor neuropathy type II; EV: empty vector; FRA: filter retardation assay; fs: frameshift; HSPA/HSP70: heat shock protein family A (Hsp70); HSPB1/Hsp27: heat shock protein family B (small) member 1; HSPB8/Hsp22: heat shock protein family B (small) member 8; HTT: huntingtin; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MD: molecular dynamics; MTOC: microtubule organizing center; MYH: myosin heavy chain; MYOG: myogenin; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; NSC34: Neuroblastoma X Spinal Cord 34; OPTN: optineurin; polyQ: polyglutamine; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1: Tax1 binding protein 1; TUBA: tubulin alpha; WT: wild-type.

Exploration of Novel Scaffolds Targeting Cytochrome b of Pyricularia oryzae.

The fulfilment of the European "Farm to Fork" strategy requires a drastic reduction in the use of "at risk" synthetic pesticides; this exposes vulnerable agricultural sectors-among which is the European risiculture-to the lack of efficient means for the management of devastating diseases, thus endangering food security. Therefore, novel scaffolds need to be identified for the synthesis of new and more environmentally friendly fungicides. In the present work, we employed our previously developed 3D model of P. oryzae cytochrome bc1 (cyt bc1) complex to perform a high-throughput virtual screening of two commercially available compound libraries. Three chemotypes were selected, from which a small collection of differently substituted analogues was designed and synthesized. The compounds were tested as inhibitors of the cyt bc1 enzyme function and the mycelium growth of both strobilurin-sensitive (WT) and -resistant (RES) P. oryzae strains. This pipeline has permitted the identification of thirteen compounds active against the RES cyt bc1 and five compounds that inhibited the WT cyt bc1 function while inhibiting the fungal mycelia only minimally. Serendipitously, among the studied compounds we identified a new chemotype that is able to efficiently inhibit the mycelium growth of WT and RES strains by ca. 60%, without inhibiting the cyt bc1 enzymatic function, suggesting a different mechanism of action.

In silico investigation on structure-function relationship of members belonging to the human SLC52 transporter family.

Riboflavin is an essential water-soluble vitamin that needs to be provided through the diet because of the conversion into flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), important cofactors in hundreds of flavoenzymes. The adsorption and distribution of riboflavin is mediated by transmembrane transporters of the SLC52 family, namely RFVT1-3, whose mutations are mainly associated with two diseases, MADD and the Brown-Vialetto-Van Laere syndrome. Interest in RFVTs as pharmacological targets has increased in the last few years due to their overexpression in several cancer cells, which can be exploited both by blocking the uptake of riboflavin into the cancerous cells, and by performing cancer targeted delivery of drugs with a high affinity for RFVTs. In this work, we propose three-dimensional structural models for all three human riboflavin transporters obtained by state-of-the-art artificial intelligence-based methods, which were then further refined with molecular dynamics simulations. Furthermore, two of the most notable mutations concerning RFVT2 and RFVT3 (W31S and N21S, respectively) were investigated studying the interactions between the wild-type and mutated transporters with riboflavin.

Molecular Modelling of NONO and SFPQ Dimerization Process and RNA Recognition Mechanism.

NONO and SFPQ are involved in multiple nuclear processes (e.g., pre-mRNA splicing, DNA repair, and transcriptional regulation). These proteins, along with NEAT1, enable paraspeckle formation, thus promoting multiple myeloma cell survival. In this paper, we investigate NONO and SFPQ dimer stability, highlighting the hetero- and homodimer structural differences, and model their interactions with RNA, simulating their binding to a polyG probe mimicking NEAT1guanine-rich regions. We demonstrated in silico that NONO::SFPQ heterodimerization is a more favorable process than homodimer formation. We also show that NONO and SFPQ RRM2 subunits are primarily required for protein-protein interactions with the other DBHS protomer. Simulation of RNA binding to NONO and SFPQ, beside validating RRM1 RNP signature importance, highlighted the role of ß2 and ß4 strand residues for RNA specific recognition. Moreover, we demonstrated the role of the NOPS region and other protomer's RRM2 ß2/ß3 loop in strengthening the interaction with RNA. Our results, having deepened RNA and DBHS dimer interactions, could contribute to the design of small molecules to modulate the activity of these proteins. RNA-mimetics, able to selectively bind to NONO and/or SFPQ RNA-recognition site, could impair paraspeckle formation, thus representing a first step towards the discovery of drugs for multiple myeloma treatment.

Functional Heterodimerization between the G Protein-Coupled Receptor GPR17 and the Chemokine Receptors 2 and 4: New Evidence.

GPR17, a G protein-coupled receptor, is a pivotal regulator of myelination. Its endogenous ligands trigger receptor desensitization and downregulation allowing oligodendrocyte terminal maturation. In addition to its endogenous agonists, GPR17 could be promiscuously activated by pro-inflammatory oxysterols and chemokines released at demyelinating lesions. Herein, the chemokine receptors CXCR2 and CXCR4 were selected to perform both in silico modelling and in vitro experiments to establish their structural and functional interactions with GPR17. The relative propensity of GPR17 and CXCR2 or CXCR4 to form homo- and hetero-dimers was assessed by homology modelling and molecular dynamics (MD) simulations, and co-immunoprecipitation and immunoenzymatic assay. The interaction between chemokine receptors and GPR17 was investigated by determining receptor-mediated modulation of intracellular cyclic adenosine monophosphate (cAMP). Our data show the GPR17 association with CXCR2 or CXCR4 and the negative regulation of these interactions by CXCR agonists or antagonists. Moreover, GPR17 and CXCR2 heterodimers can functionally influence each other. In contrast, CXCR4 can influence GPR17 functionality, but not vice versa. According to MD simulations, all the dimers reached conformational stability and negative formation energy, confirming the experimental observations. The cross-talk between these receptors could play a role in the development of the neuroinflammatory milieu associated with demyelinating events.

rHDL modeling and the anchoring mechanism of LCAT activation.

Lecithin:cholesterol-acyl transferase (LCAT) plays a major role in cholesterol metabolism as it is the only extracellular enzyme able to esterify cholesterol. LCAT activity is required for lipoprotein remodeling and, most specifically, for the growth and maturation of HDLs. In fact, genetic alterations affecting LCAT functionality may cause a severe reduction in plasma levels of HDL-cholesterol with important clinical consequences. Although several hypotheses were formulated, the exact molecular recognition mechanism between LCAT and HDLs is still unknown. We employed a combination of structural bioinformatics procedures to deepen the insights into the HDL-LCAT interplay that promotes LCAT activation and cholesterol esterification. We have generated a data-driven model of reconstituted HDL (rHDL) and studied the dynamics of an assembled rHDL::LCAT supramolecular complex, pinpointing the conformational changes originating from the interaction between LCAT and apolipoprotein A-I (apoA-I) that are necessary for LCAT activation. Specifically, we propose a mechanism in which the anchoring of LCAT lid to apoA-I helices allows the formation of a hydrophobic hood that expands the LCAT active site and shields it from the solvent, allowing the enzyme to process large hydrophobic substrates.

Structural insights into the desymmetrization of bulky 1,2-dicarbonyls through enzymatic monoreduction.

Benzil reductases are dehydrogenases preferentially active on aromatic 1,2-diketones, but the reasons for this peculiar substrate recognition have not yet been clarified. The benzil reductase (KRED1-Pglu) from the non-conventional yeast Pichia glucozyma showed excellent activity and stereoselectivity in the monoreduction of space-demanding aromatic 1,2-dicarbonyls, making this enzyme attractive as biocatalyst in organic chemistry. Structural insights into the stereoselective monoreduction of 1,2-diketones catalyzed by KRED1-Pglu were investigated starting from its 1.77 Å resolution crystal structure, followed by QM and classical calculations; this study allowed for the identification and characterization of the KRED1-Pglu reactive site. Once identified the recognition elements involved in the stereoselective desymmetrization of bulky 1,2-dicarbonyls mediated by KRED1-Pglu, a mechanism was proposed together with an in silico prediction of substrates reactivity.

Activation of Naturally Occurring Lecithin:Cholesterol Acyltransferase Mutants by a Novel Activator Compound.

Lecithin:cholesterol acyltransferase (LCAT) is a unique plasma enzyme able to esterify cholesterol, and it plays an important role in HDL maturation and promotion of reverse cholesterol transport. Familial LCAT deficiency (FLD; OMIM number 245900) is a rare recessive disease that results from loss-of-function mutations in the LCAT gene and has no cure. In this study, we assessed the in vitro efficacy of a novel small-molecule LCAT activator. Cholesterol esterification rate (CER) and LCAT activity were tested in plasma from six controls and five FLD homozygous carriers of various LCAT mutations at different doses of the compound (0.1, 1, and 10 µg/ml). In control plasma, the compound significantly increased both CER (P < 0.001) and LCAT activity (P = 0.007) in a dose-dependent manner. Both CER and LCAT activity increased by 4- to 5-fold, reaching maximum activation at the dose of 1 µg/ml. Interestingly, Daiichi Sankyo compound produced an increase in CER in two of the five tested LCAT mutants (Leu372--Arg and Val309--Met), while LCAT activity increased in three LCAT mutants (Arg147--Trp, Thr274--Ile and Leu372--Arg); mutant Pro254--Ser was not activated at any of the tested doses. The present findings form the basis for personalized therapeutic interventions in FLD carriers and support the potential LCAT activation in secondary LCAT defects. SIGNIFICANCE STATEMENT: We characterized the pharmacology of a novel small-molecule LCAT activator in vitro on a subset of naturally occurring LCAT mutants. Our findings form the basis for personalized therapeutic interventions for familial LCAT deficiency carriers, who can face severe complications and for whom no cure exists.

SLC6A14, a Pivotal Actor on Cancer Stage: When Function Meets Structure.

SLC6A14 (ATB0,+) is a sodium- and chloride-dependent neutral and dibasic amino acid transporter that regulates the distribution of amino acids across cell membranes. The transporter is overexpressed in many human cancers characterized by an increased demand for amino acids; as such, it was recently acknowledged as a novel target for cancer therapy. The knowledge on the molecular mechanism of SLC6A14 transport is still limited, but some elegant studies on related transporters report the involvement of the 12 transmembrane α-helices in the transport mechanism, and describe structural rearrangements mediated by electrostatic interactions with some pivotal gating residues. In the present work, we constructed a SLC6A14 model in outward-facing conformation via homology modeling and used molecular dynamics simulations to predict amino acid residues critical for substrate recognition and translocation. We docked the proteinogenic amino acids and other known substrates in the SLC6A14 binding site to study both gating regions and the exposed residues involved in transport. Interestingly, some of these residues correspond to those previously identified in other LeuT-fold transporters; however, we could also identify a novel relevant residue with such function. For the first time, by combined approaches of molecular docking and molecular dynamics simulations, we highlight the potential role of these residues in neutral amino acid transport. This novel information unravels new aspects of the human SLC6A14 structure-function relationship and may have important outcomes for cancer treatment through the design of novel inhibitors of SLC6A14-mediated transport.

What if? Mouse proteomics after gene inactivation.

The complex interactions among proteins and of proteins with small molecular weight protein ligands are overturned every time one of the components of the network is missing. For study purposes, animal models lacking one protein are obtained by experimental manipulation of the genome: in the knocking out approach, a gene is altered through the insertion of an artificial DNA sequence, which halts the transcription-translation sequence of events. In this review we have compiled the research papers that analyze the effects of knocking out individual genes on the proteomes of various tissues/organs throughout the body. We have gathered and organized all the available evidence and then compared the proteomic data in order to stress the context-specificity of the outcome every time two or more organs were investigated in the same KO mice. Finally, in a symmetrical approach to the above, we surveyed whether there is any obvious overlap among the effects of different KO on the same organ, marking affection of general pathways or lacking specificity of the gene targeting. Specific attention was put on the possible involvement of cellular stress markers.

Strategic single point mutation yields a solvent- and salt-stable transaminase from Virgibacillus sp. in soluble form.

A new transaminase (VbTA) was identified from the genome of the halotolerant marine bacterium Virgibacillus 21D. Following heterologous expression in Escherichia coli, it was located entirely in the insoluble fraction. After a single mutation, identified via sequence homology analyses, the VbTA T16F mutant was successfully expressed in soluble form and characterised. VbTA T16F showed high stability towards polar organic solvents and salt exposure, accepting mainly hydrophobic aromatic amine and carbonyl substrates. The 2.0 Å resolution crystal structure of VbTA T16F is here reported, and together with computational calculations, revealed that this mutation is crucial for correct dimerisation and thus correct folding, leading to soluble protein expression.

In silico Description of LAT1 Transport Mechanism at an Atomistic Level.

The molecular mechanism of transport mediated by LAT1, a sodium-independent antiporter of large neutral amino acids, was investigated through in silico procedures, specifically making reference to two transported substrates, tyrosine (Tyr) and leucine methyl ester (LME), and to 3,5-diiodo-L-tyrosine (DIT), a well-known LAT1 inhibitor. Two models of the transporter were built by comparative modeling, with LAT1 either in an outward-facing (OF) or in an inward-facing (IF) conformation, based, respectively, on the crystal structure of AdiC and of GadC. As frequently classic Molecular Dynamics (MD) fails to monitor large-scale conformational transitions within a reasonable simulated time, the OF structure was equilibrated for 150 ns then processed through targeted MD (tMD). During this procedure, an elastic force pulled the OF structure to the IF structure and induced, at the same time, substrates/inhibitor to move through the transport channel. This elastic force was modulated by a spring constant (k) value; by decreasing its value from 100 to 70, it was possible to comparatively account for the propensity for transport of the three tested molecules. In line with our expectations, during the tMD simulations, Tyr and LME behaved as substrates, moving down the transport channel, or most of it, for all k values. On the contrary, DIT behaved as an inhibitor, being (almost) transported across the channel only at the highest k value (100). During their transit through the channel, Tyr and LME interacted with specific amino acids (first with Phe252 then with Thr345, Arg348, Tyr259, and Phe262); this suggests that a primary as well as a putative secondary gate may contribute to the transport of substrates. Quite on the opposite, DIT appeared to establish only transient interactions with side chains lining the external part of the transport channel. Our tMD simulations could thus efficiently discriminate between two transported substrates and one inhibitor, and therefore can be proposed as a benchmark for developing novel LAT1 inhibitors of pharmacological interest.