Partner Molecules Research Articles

Diosgenin upregulates axonal guidance partner molecules, Galectin-1 and Secernin-1

Galectin-1, a β-galactosides-binding protein, is widely expressed in various tissues and exhibits diverse biological activities. We previously obtained following findings; 1) Diosgenin, a steroid sapogenin, promoted axonal regeneration in the brain and recovered memory deficits in a model of Alzheimer’s disease (AD), 5XFAD mouse; 2) Neuron-specific overexpression of Galectin-1 protein in the hippocampus recovered memory impairment and promoted axonal regeneration in the brain in 5XFAD mice; 3) Secernin-1, a counterpart and axonal guidance molecule for Galectin-1-expressing axons, was secreted from the prefrontal cortical neurons to promote axonal guidance from the hippocampus to the prefrontal cortex. However, it has never been elucidated that diosgenin signaling increase Galectin-1 and Secernin-1 or not. Here, we found that diosgenin treatment upregulated the protein level of Galectin-1 in the hippocampus both in primary cultured neurons and in 5XFAD mouse brains. In addition, diosgenin-induced upregulation of Galectin-1 was diminished by treatment of a neutralizing antibody of 1,25D3-membrane-associated rapid response steroid-binding receptor (1,25D3-MARRS), a direct binding receptor for diosgenin. Importantly, knockdown of Galectin-1 in hippocampal neurons inhibited axonal growth activity of diosgenin. Furthermore, the expression level of Secernin-1 was also increased in prefrontal cortical neurons by administration of diosgenin to 5XFAD mice. These findings suggest that diosgenin is a suitable compound to facilitate Galectin-1-Secernin-1-mediated axonal growth in AD brains.

HIF-1α and MIF enhance neutrophil-driven type 3 immunity and chondrogenesis in a murine spondyloarthritis model.

The hallmarks of spondyloarthritis (SpA) are type 3 immunity-driven inflammation and new bone formation (NBF). Macrophage migration inhibitory factor (MIF) was found to be a key driver of the pathogenesis of SpA by amplifying type 3 immunity, yet MIF-interacting molecules and networks remain elusive. Herein, we identified hypoxia-inducible factor-1 alpha (HIF1A) as an interacting partner molecule of MIF that drives SpA pathologies, including inflammation and NBF. HIF1A expression was increased in the joint tissues and synovial fluid of SpA patients and curdlan-injected SKG (curdlan-SKG) mice compared to the respective controls. Under hypoxic conditions in which HIF1A was stabilized, human and mouse neutrophils exhibited substantially increased expression of MIF and IL-23, an upstream type 3 immunity-related cytokine. Similar to MIF, systemic overexpression of IL-23 induced SpA pathology in SKG mice, while the injection of a HIF1A-selectiveinhibitor (PX-478) into curdlan-SKG mice prevented or attenuated SpA pathology, as indicated by a marked reduction in the expression of MIF and IL-23. Furthermore, genetic deletion of MIF or HIF1A inhibition with PX-478 in IL-23-overexpressing SKG mice did not induce evident arthritis or NBF, despite the presence of psoriasis-like dermatitis and blepharitis. We also found that MIF- and IL-23-expressing neutrophils infiltrated areas of the NBF in curdlan-SKG mice. These neutrophils potentially increased chondrogenesis and cell proliferation via the upregulation of STAT3 in periosteal cells and ligamental cells during endochondral ossification. Together, these results provide supporting evidence for an MIF/HIF1A regulatory network, and inhibition of HIF1A may be a novel therapeutic approach for SpA by suppressing type 3 immunity-mediated inflammation and NBF.

Halogen Bond via an Electrophilic π-Hole on Halogen in Molecules: Does It Exist?

This study reveals a new non-covalent interaction called a π-hole halogen bond, which is directional and potentially non-linear compared to its sister analog (σ-hole halogen bond). A π-hole is shown here to be observed on the surface of halogen in halogenated molecules, which can be tempered to display the aptness to form a π-hole halogen bond with a series of electron density-rich sites (Lewis bases) hosted individually by 32 other partner molecules. The [MP2/aug-cc-pVTZ] level characteristics of the π-hole halogen bonds in 33 binary complexes obtained from the charge density approaches (quantum theory of intramolecular atoms, molecular electrostatic surface potential, independent gradient model (IGM-δginter)), intermolecular geometries and energies, and second-order hyperconjugative charge transfer analyses are discussed, which are similar to other non-covalent interactions. That a π-hole can be observed on halogen in halogenated molecules is substantiated by experimentally reported crystals documented in the Cambridge Crystal Structure Database. The importance of the π-hole halogen bond in the design and growth of chemical systems in synthetic chemistry, crystallography, and crystal engineering is yet to be fully explicated.

EBP50 Depletion and Nuclear β-Catenin Accumulation Engender Aggressive Behavior of Colorectal Carcinoma through Induction of Tumor Budding.

Ezin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a scaffold protein that interacts with several partner molecules including β-catenin. Here, we examined the crosstalk between EBP50 and nuclear catenin during colorectal carcinoma (CRC) progression. In clinical samples, there were no correlations between the subcellular location of EBP50 and any clinicopathological factors. However, EBP50 expression was significantly lower specifically in the outer areas of tumor lesions, in regions where tumor budding (BD) was observed. Low EBP50 expression was also significantly associated with several unfavorable prognostic factors, suggesting that EBP50 depletion rather than its overexpression or subcellular distribution plays an important role in CRC progression. In CRC cell lines, knockout of EBP50 induced epithelial-mesenchymal transition (EMT)-like features, decreased proliferation, accelerated migration capability, and stabilized nuclear β-catenin due to disruption of the interaction between EBP50 and β-catenin at the plasma membrane. In addition, Slug expression was significantly higher in outer lesions, particularly in BD areas, and was positively correlated with nuclear β-catenin status, consistent with β-catenin-driven transactivation of the Slug promoter. Together, our data suggest that EBP50 depletion releases β-catenin from the plasma membrane in outer tumor lesions, allowing β-catenin to accumulate and translocate to the nucleus, where it transactivates the Slug gene to promote EMT. This in turn triggers tumor budding and contributes to the progression of CRC to a more aggressive phase.

Probing excited state 1Hα chemical shifts in intrinsically disordered proteins with a triple resonance-based CEST experiment: Application to a disorder-to-order switch.

Over 40% of eukaryotic proteomes and 15% of bacterial proteomes are predicted to be intrinsically disordered based on their amino acid sequence. Intrinsically disordered proteins (IDPs) exist as heterogeneous ensembles of interconverting conformations and pose a challenge to the structure-function paradigm by apparently functioning without possessing stable structural elements. IDPs play a prominent role in biological processes involving extensive intermolecular interaction networks and their inherently dynamic nature facilitates their promiscuous interaction with multiple structurally diverse partner molecules. NMR spectroscopy has made pivotal contributions to our understanding of IDPs because of its unique ability to characterize heterogeneity at atomic resolution. NMR methods such as Chemical Exchange Saturation Transfer (CEST) and relaxation dispersion have enabled the detection of 'invisible' excited states in biomolecules which are transiently and sparsely populated, yet central for function. Here, we develop a 1Hα CEST pulse sequence which overcomes the resonance overlap problem in the 1Hα-13Cα plane of IDPs by taking advantage of the superior resolution in the 1H-15N correlation spectrum. In this sequence, magnetization is transferred after 1H CEST using a triple resonance coherence transfer pathway from 1Hα (i) to 1HN(i+1) during which the 15N(t1) and 1HN(t2) are frequency labelled. This approach is integrated with spin state-selective CEST for eliminating spurious dips in CEST profiles resulting from dipolar cross-relaxation. We apply this sequence to determine the excited state 1Hα chemical shifts of the intrinsically disordered DNA binding domain (CytRN) of the bacterial cytidine repressor (CytR), which transiently acquires a functional globally folded conformation. The structure of the excited state, calculated using 1Hα chemical shifts in conjunction with other excited state NMR restraints, is a three-helix bundle incorporating a helix-turn-helix motif that is vital for binding DNA.

CoMemMoRFPred: Sequence-based Prediction of MemMoRFs by Combining Predictors of Intrinsic Disorder, MoRFs and Disordered Lipid-binding Regions

Molecular recognition features (MoRFs) are a commonly occurring type of intrinsically disordered regions (IDRs) that undergo disorder-to-order transition upon binding to partner molecules. We focus on recently characterized and functionally important membrane-binding MoRFs (MemMoRFs). Motivated by the lack of computational tools that predict MemMoRFs, we use a dataset of experimentally annotated MemMoRFs to conceptualize, design, evaluate and release an accurate sequence-based predictor. We rely on state-of-the-art tools that predict residues that possess key characteristics of MemMoRFs, such as intrinsic disorder, disorder-to-order transition and lipid-binding. We identify and combine results from three tools that include flDPnn for the disorder prediction, DisoLipPred for the prediction of disordered lipid-binding regions, and MoRFCHiBiLight for the prediction of disorder-to-order transitioning protein binding regions. Our empirical analysis demonstrates that combining results produced by these three methods generates accurate predictions of MemMoRFs. We also show that use of a smoothing operator produces predictions that closely mimic the number and sizes of the native MemMoRF regions. The resulting CoMemMoRFPred method is available as an easy-to-use webserver at http://biomine.cs.vcu.edu/servers/CoMemMoRFPred. This tool will aid future studies of MemMoRFs in the context of exploring their abundance, cellular functions, and roles in pathologic phenomena.

DichroIDP: a method for analyses of intrinsically disordered proteins using circular dichroism spectroscopy

Intrinsically disordered proteins (IDPs) are comprised of significant numbers of residues that form neither helix, sheet, nor any other canonical type of secondary structure. They play important roles in a broad range of biological processes, such as molecular recognition and signalling, largely due to their chameleon-like ability to change structure from unordered when free in solution to ordered when bound to partner molecules. Circular dichroism (CD) spectroscopy is a widely-used method for characterising protein secondary structures, but analyses of IDPs using CD spectroscopy have suffered because the methods and reference datasets used for the empirical determination of secondary structures do not contain adequate representations of unordered structures. This work describes the creation, validation and testing of a standalone Windows-based application, DichroIDP, and a new reference dataset, IDP175, which is suitable for analyses of proteins containing significant amounts of disordered structure. DichroIDP enables secondary structure determinations of IDPs and proteins containing intrinsically disordered regions.

Destruction of Octa(4-tert-butylphenyl)tetrapyrazinoporphyrazine Under the Influence of Nitrogen-Containing Organic Bases in Dimethylsulfoxide

A study is performed of the state of octa(4-tert-butylphenyl)tetrapyrazinoporphyrazine in dimethyl sulfoxide. It is found that the acid–base interaction of partner molecules creates a time-stable complex with proton transfer. It is shown that adding morpholine, piperidine, n-butylamine, and diethylamine to dimethyl sulfoxide results in destruction of this complex, in contrast to adding pyridine, 2-methylpyridine, tert-butylamine, or tri-n-butylamine. The effect the proton-acceptor ability and the spatial structure of the base have on the decomposition of the tetrapyrazinoporphyrazine macrocycle is considered.

Divergent Sex-Specific Effects of Toll-like Receptor 4 and Cluster of Differentiation 14 in Dahl Salt-Sensitive Hypertension

Renal inflammation accompanies hypertension and renal damage in Dahl Salt-Sensitive rats fed a high salt diet (HS, 4.0% NaCl). Via a transcriptomics approach, we previously found cluster of differentiation 14 (CD14) and toll-like receptor 4 (TLR4), partner molecules involved in innate immune system activation, to be upregulated in renal medullary tissue of Dahl SS rats after a HS challenge. A subsequent study demonstrated that both CD14 and TLR4 are upregulated in macrophages infiltrating the kidneys of Dahl SS fed HS. Interestingly, CD14 deletion on the Dahl SS background confers an amplification of hypertension and renal damage in females but has no effect in males. Given this finding, we examined sex-specific effects of TLR4 deletion in the pathogenesis of Dahl SS hypertension. The current study tested the hypothesis that TLR4 knockout differentially modulates salt-sensitivity in males versus females. A mutation was generated in the TLR4 gene on the Dahl SS background using CRISPR-Cas9 technology to create a 1-basepair frameshift mutation resulting in a premature stop codon. SS TLR4+/+ and SS TLR4-/- males (n=10) and females (n=6), maintained on a low salt chow (LS, 0.4% NaCl), underwent telemeter implantation at approximately 7 weeks of age for continuous monitoring of blood pressure. Following recovery and baseline blood pressure recordings, animals were switched to a HS chow for 3 weeks. Urine samples were collected once during the LS period and weekly throughout the HS challenge. Kidneys were flushed and harvested for analysis. Mean arterial pressure (MAP) was not different between the groups during the baseline period (117.3 ± 1, 116.9 ± 1, 117.3 ± 1, 116.9 ± 2 mmHg). Throughout the HS challenge, MAP was significantly reduced in the male SS TLR4-/- compared to male SS TLR4+/+ (day 19: 150.8 ± 4 vs. 162.5 ± 5 mmHg, p=0.010), but MAP was unchanged in female SS TLR4-/- compared to SSTLR4+/+ (151.6 ± 4 vs 155.3 ± 4 mmHg, p=0.554). Similarly, urinary albumin excretion was reduced in the male SS TLR4-/- versus SS TLR4+/+ (170.3 ± 27 vs 321.3 ± 63 mg/day, p=0.023), indicative of reduced renal injury in these animals. Albuminuria was not observed to be different between female SS TLR4-/- and SS TLR4+/+ (149.7 ± 24 vs 145.9 ± 14, p=0.448). At the end of the HS challenge, immune cells were isolated from the kidneys of these animals and characterized by flow cytometric analysis. We observed no difference in either the circulating or infiltrating immune cell profile between wildtypes and knockouts. These results indicate a male-specific effect of TLR4 deletion and suggest that TLR4 promotes salt-sensitive hypertension and renal damage in males. This finding contrasts with the effect of TLR4’s partner molecule, CD14, which attenuates SS hypertension in females. Further studies are needed to elucidate the molecular mechanisms of TLR4/CD14 activation and signaling during salt-sensitive hypertension. HL161231 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Obesity Is Associated with Distorted Proteoglycan Expression in Adipose Tissue.

Proteoglycans are central components of the extracellular matrix (ECM) and binding partners for inflammatory chemokines. Morphological differences in the ECM and increased inflammation are prominent features of the white adipose tissues in patients with obesity. The impact of obesity and weight loss on the expression of specific proteoglycans in adipose tissue is not well known. This study aimed to investigate the relationship between adiposity and proteoglycan expression. We analyzed transcriptomic data from two human bariatric surgery cohorts. In addition, RT-qPCR was performed on adipose tissues from female and male mice fed a high-fat diet. Both visceral and subcutaneous adipose tissue depots were analyzed. Adipose mRNA expression of specific proteoglycans, proteoglycan biosynthetic enzymes, proteoglycan partner molecules, and other ECM-related proteins were altered in both human cohorts. We consistently observed more profound alterations in gene expression of ECM targets in the visceral adipose tissues after surgery (among others VCAN (p = 0.000309), OGN (p = 0.000976), GPC4 (p = 0.00525), COL1A1 (p = 0.00221)). Further, gene analyses in mice revealed sex differences in these two tissue compartments in obese mice. We suggest that adipose tissue repair is still in progress long after surgery, which may reflect challenges in remodeling increased adipose tissues. This study can provide the basis for more mechanistic studies on the role of proteoglycans in adipose tissues in obesity.

Reduction of CO2 with Hydrated Electrons: Ab Initio Computational Studies for Finite-Size Cluster Models.

In the present work, the mechanisms of the reduction of the CO2 molecule with hydrated electrons to the hydroxyl-formyl (HOCO) radical were studied with ab initio computational methods. Hydrated hydronium radicals, H3O(H2O)n (n = 0,3,6), are considered as finite-size models of the hydrated electron in liquid water. The investigation of cluster models allows the application of high-accuracy electronic-structure methods, which are not computationally feasible in condensed-phase simulations. Reaction paths and potential-energy (PE) profiles of the proton-coupled electron-transfer reaction from hydrated H3O radicals to the CO2 molecule were explored on the ground-state PE surface. The computationally efficient unrestricted second-order Møller-Plesset method is employed, and its accuracy has been carefully benchmarked in comparison with complete-active-space self-consistent-field and multi-reference second-order perturbation calculations. The results provide insights into the interplay of electron transfer from the diffuse Rydberg-type unpaired electron of H3O to the CO2 molecule, the contraction of the electron cloud by the re-hybridization of the carbon atom of CO2, and proton transfer from the nearest water molecule to the CO2- anion, followed by Grotthus-type proton rearrangements to form stable clusters. Starting from local energy minima of hydrogen-bonded CO2-H3O(H2O)n complexes, the reaction to form HOCO-(H2O)n+1 complexes is exothermic by about 1.3 eV (125 kJ/mol). The reaction is barrier controlled with a barrier of the order of a few tenths of an electron volt, depending on size and conformation of the water cluster. This barrier is at least an order of magnitude lower than the barrier of the reaction of CO2 with any closed-shell partner molecule. The HOCO radicals can recombine by H-atom transfer (disproportionation), resulting in formic acid or a dihydroxycarbene product, as well as by the formation of a C-C bond, resulting in oxalic acid. The strong exothermicity of these radical-radical recombination reactions likely results in the fragmentation of the closed-shell products formic acid and oxalic acid, which explains the strong specificity for CO formation observed in recent experiments of Hamers and co-workers.

The oncogenic circular RNA circ_63706 is a potential therapeutic target in sonic hedgehog-subtype childhood medulloblastomas

Medulloblastoma (MB) develops through various genetic, epigenetic, and non-coding (nc) RNA-related mechanisms, but the roles played by ncRNAs, particularly circular RNAs (circRNAs), remain poorly defined. CircRNAs are increasingly recognized as stable non-coding RNA therapeutic targets in many cancers, but little is known about their function in MBs. To determine medulloblastoma subgroup-specific circRNAs, publicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify circRNAs that differentiate between MB subgroups. circ_63706 was identified as sonic hedgehog (SHH) group-specific, with its expression confirmed by RNA-FISH analysis in clinical tissue samples. The oncogenic function of circ_63706 was characterized in vitro and in vivo. Further, circ_63706-depleted cells were subjected to RNA-seq and lipid profiling to identify its molecular function. Finally, we mapped the circ_63706 secondary structure using an advanced random forest classification model and modeled a 3D structure to identify its interacting miRNA partner molecules. Circ_63706 regulates independently of the host coding gene pericentrin (PCNT), and its expression is specific to the SHH subgroup. circ_63706-deleted cells implanted into mice produced smaller tumors, and mice lived longer than parental cell implants. At the molecular level, circ_63706-deleted cells elevated total ceramide and oxidized lipids and reduced total triglyceride. Our study implicates a novel oncogenic circular RNA in the SHH medulloblastoma subgroup and establishes its molecular function and potential as a future therapeutic target.

Abstract P2-11-29: The Expression of Claudin-10 in relationship with Her family members in patients with breast cancer

Abstract Introduction. Claudin-10 (CLDN10), a member of the Claudin tight junction (TJ) protein family, was initially known as Oligodendrocyte-Specific Protein-Like (OSP-L). The distribution of CLDN10 in the body is not ubiquitous and is seen more abundantly in kidney and brain and at modest levels in mammary tissues. Limited information suggests that in cells where CLDN10 is expressed, it interacts with other family members including CLDN-11, 12, 16, 17 and the subcoat zonula occludens (ZO-1 and ZO-3) to regulate TJ functions. The role played by CLDN10 in cancer is not clear but there are suggestions that it is a favourable indicator for disease progression in kidney and gastric cancers but an adverse indictor in hormone related cancers such as thyroid cancer. The role of CLDN10 in breast cancer is otherwise not known. We have investigated the expression profile and in particular in its relationship with hormone receptor status. Methods. We carried out CLDN10 transcript and protein analyses by way of quantitative PCR and immunohistochemistry on an established cohort of fresh frozen mammary tissues and breast cancer tissues. Expression was analysed via QPCR against the staging, histology, clinical outcome, hormone status including ER and Her family members, and also its known interactive TJ molecules available to our database. Results. Compared with its interactive TL partner molecules, CLDN10 was expressed at relatively low levels in mammary tissues. There was no significant difference in CLDN10 transcript levels between normal tissues and tumour tissues. Patients with high levels of CLDN10 in breast tumours had significantly shorter overall survival (OS) (p=0.041) and disease free survival (DFS) (p=0.026). In our cohort, we found a significant correlation between levels of CLDN10 and Her-1/EGFR (r=0.201, p=0.026) and Her3 (r=0.935, p< 0.0001), but not with Her-2 nor with Her-4. It was also surprised to find that CLDN10 did not correlated with the ZO-1, ZO-3, CLDN11, 12, 14, and 17. We then identified a subgroup of patients with low levels of CLDN10 and their overall survival (OS) and disease free survival (DFS) which were significantly correlated with Her-1/Her-3 expression status (p< 0.0001 for both OS and DFS). CLDN10, amongst the clinical, pathological and hormone receptor status, is an independent prognostic factor for DFS (p=0.008, Hazard Ratio (HR) =4.228 (95% CI 1.449-12.342)) and for OS (p=0.025, HR=3.917 (95% CI 1.187-12.924)). The predictive power for triple negative breast cancer (TNBC) and non-TNBC by CLDN10 otherwise remained similar and not statistically significant. It was noted that stratification by ER and Her-2 did not contribute to the value of independency. Conclusion. CLDN10, although expressed at relative lower levels in breast cancer, is a contributing factor to the survival of the patients. Together with the status of EGFR and Her-3, it makes an independent prognostic factor for both the overall survival and disease free survival of the patients. Citation Format: Xinguo Zhuang, Wen G. Jiang, Fiona Ruge, Eleri Davies, Bing Xu, Tracey A. Martin. The Expression of Claudin-10 in relationship with Her family members in patients with breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-29.

Synthesis and Self-Assembly of β-Octa[(4-Diethoxyphosphoryl)phenyl]porphyrins.

The β-substituted porphyrinoids commonly used to form functional assembled systems in nature yet are still scarcely used in material chemistry probably due to the laborious synthesis of these compounds. In this work, β-octa[(4-diethoxyphosphoryl)phenyl]porphyrin (2HOPPP) and its metal (Zn(II), Cd(II), Cu(II), and Ni(II)) complexes were prepared in good yields. These highly soluble chromophores were characterized in solution using spectroscopic (NMR, UV-vis, fluorescence), electrochemical, and spectroelectrochemical methods. Attachment of the electron-deficient residue (ArP(O)(OEt)2) to the porphyrin macrocycle leads to easier reductions and harder oxidations of the macrocycle for all complexes studied as compared to corresponding meso-tetra[4-(diethoxyphosphoryl)phenyl]porphyrin derivatives reported previously. We demonstrated that the strong electron-deficient character of the MOPPP porphyrins results principally from the increase in the number of electron-withdrawing groups at the periphery of the tetrapyrrolic macrocycle. Electron-deficient porphyrins are highly required in supramolecular and material chemistry in part due to their ability to form supramolecular assemblies via the coordination of axial ligands to the central metal atom. According to single-crystal X-ray data, ZnOPPP forms in the crystalline phase dimers in which each of the two tetrapyrrolic macrocycles is connected through an unusual combination of hydrogen bonding of two phosphoryl groups and the water molecules axially coordinated to the zinc atom of the partner molecule. The involvement of water molecules in porphyrin binding allows for an increase of distance between two porphyrin mean N4 planes, up to 4.478 Å. The offset of phosphoryl groups attached to the macrocycle through a 1,4-phenylene spacer withdraws the whole porphyrin macrocycle of one molecule from spatial overlap with the macrocycle of a partner molecule and increases the Zn-Zn distance up to 10.372 Å. This still unknown type of porphyrin dimers allows one to get deeper insights into the organization of naturally occurring tetrapyrrolic macrocycles. ZnOPPP also forms a labile dimeric complex in 5.3 × 10-7-5.8 × 10-5 M chloroform solutions. In contrast, other complexes prepared in this work exist as monomeric species under these experimental conditions. The self-association constant of ZnOPPP has been determined by electronic absorption spectroscopy.

Classification of proteins inducing liquid-liquid phase separation: sequential, structural and functional characterization.

Liquid-liquid phase separation (LLPS) within the cell can form biological condensates, which are increasingly recognized to play important roles in various biological processes. Most proteins involved in LLPS are known to be intrinsically disordered proteins containing intrinsically disordered regions (IDRs) with low complexity regions (LCRs). The proteins driving LLPS were selected from databases of LLPS-related proteins and then classified into three classes according to the components in the condensates. Through in silico analyses, we found that proteins in the homo class, those that induce LLPS without partner molecules, have different IDRs and LCRs compared with the reference proteome. In contrast, proteins in the other classes, those that induce LLPS with partner proteins (the hetero class) or nucleic acids (the mixed class), did not show a clear difference to the reference proteome in IDRs and LCRs. The hetero-class proteins contained structural domains to serve protein-protein interactions, and the mixed-class ones had the structural domains associated with nucleic acids. These results suggest that IDRs in the homo-class proteins have unique IDRs, which provide multivalent interaction sites required for LLPS, whereas the hetero- and mixed-class proteins can induce LLPS through the combination of the interaction among LCRs, structural domains and nucleic acids.

Explicit versus implicit consideration of binding partners in protein-protein complex to elucidate intrinsic dynamics.

The binding of many proteins to their protein partners is tightly regulated via control of their relative intrinsic dynamics during the binding process, a phenomenon which can in turn be modulated. Therefore, investigating the intrinsic dynamics of proteins is necessary to understand function in a comprehensive way. By intrinsic dynamics herein, we principally refer to the vibrational signature of a protein molecule popularly obtained from normal modes or essential modes. For normal modes, one often considers that the molecule under investigation is a collection of springs in a solvent-free or implicit-solvent medium. In the context of a protein-binding partner, the analysis of vibration of the target protein is often complicated due to molecular interaction within the complex. Generally, it is assumed that the isolated bound conformation of the target protein captures the implicit effect of the binding partner on the intrinsic dynamics, therefore suggesting that any influence of the partner molecule is also already integrated. Such an assumption allows large-scale studies of the conservation of protein flexibility. However, in cases where a partner protein directly influences the vibration of the target via critical contacts at the protein-protein interface, the above assumption falls short of providing a detailed view. In this review article, we discuss the implications of considering the dynamics of a protein in a protein-protein complex, as modelled implicitly and explicitly with methods dependent on elastic network models. We further propose how such an explicit consideration can be applied to understand critical protein-protein contacts that can be targeted in future studies.

Computational Characterisation, Homology Modelling and Structure-based Functional Annotation of Very Low-density Lipoprotein Receptor of Salmo trutta fario

The present study was designed to enlist the physiochemical and functional properties of the very low-density lipoprotein receptor of Salmo trutta fario (brown trout), and provide information about its three-dimensional structure and interaction with partner molecules using standard bioinformatic tools. VLDL receptor is a large flexible protein with a molecular weight of around 95.9 kDa, relatively unstable, and hydrophobic with a primary transmembrane helix from N-786 to C-808. All of the 70 Cysteine residues (except four) are in a disulphide bonding state. Secondary structure analysis shows that most of the protein has a predominant random coiled configuration followed by extended strands. Six Epidermal growth factor-like domains and two EGF-like-Ca2+ binding domains were predicted. The protein plays a crucially important role in various metabolic pathways including vitellogenesis in fishes. Understanding the structural and functional properties of the VLDL receptor will facilitate a better understanding of its molecular dynamics and the designing of experimental procedures.

The Scaffold Immunophilin FKBP51 Is a Phosphoprotein That Undergoes Dynamic Mitochondrial-Nuclear Shuttling

The immunophilin FKBP51 forms heterocomplexes with molecular chaperones, protein-kinases, protein-phosphatases, autophagy-related factors, and transcription factors. Like most scaffold proteins, FKBP51 can use a simple tethering mechanism to favor the efficiency of interactions with partner molecules, but it can also exert more complex allosteric controls over client factors, the immunophilin itself being a putative regulation target. One of the simplest strategies for regulating pathways and subcellular localization of proteins is phosphorylation. In this study, it is shown that scaffold immunophilin FKBP51 is resolved by resolutive electrophoresis in various phosphorylated isoforms. This was evidenced by their reactivity with specific anti-phosphoamino acid antibodies and their fade-out by treatment with alkaline phosphatase. Interestingly, stress situations such as exposure to oxidants or in vivo fasting favors FKBP51 translocation from mitochondria to the nucleus. While fasting involves phosphothreonine residues, oxidative stress involves tyrosine residues. Molecular modeling predicts the existence of potential targets located at the FK1 domain of the immunophilin. Thus, oxidative stress favors FKBP51 dephosphorylation and protein degradation by the proteasome, whereas FK506 binding protects the persistence of the post-translational modification in tyrosine, leading to FKBP51 stability under oxidative conditions. Therefore, FKBP51 is revealed as a phosphoprotein that undergoes differential phosphorylations according to the stimulus.

Molecular Systems Network Predicts Sars-Cov-2 NSP3 and Orf6 Role in COVID-19 Related Thrombotic Events

Background: Pathological advancement of SARS-CoV-2 infection entails engagement of blood related ailment including thrombosis as one of the clinical manifestations. SARS-CoV-2-Human protein-protein and protein-RNA interactomes have been explored to considerable detail. Objective: The present study is designed to develop and functionally analyze a combined molecular network of SARS-CoV-2-Human and Thrombosis to delineate candidate molecules that could subsequently be used for the prognosis and designing therapeutic interventions. Methodology: Briefly, two separate system networks were developed, one for over 500 humans proteins that have shown to interact with the viral genome and 26 different proteins encoded by the SARS-CoV-2 genome. The second network is based on the candidate genes tagged for being aberrated genetically and/or in terms of expression in thrombosis. Both networks were later combined as a singular entity after removing the redundant repetition and orphans' nodes and edges were removed by selective enrichment. The network can then be stratified in different modules primarily based on the promiscuity of the nodes. Complete network and each module were then assessed for in betweenness and shortest path length of edges. Results: The total interactome contain over 572 nodes sharing a total of 6,283 edges, suggesting substantial cross talk between genes and their encoded protein. The complete systems network comprised of total 22 modules with varying level of intermolecular interactions. Nearly half of the edges (n=246) are stationed with different modules. Out of 60 leading candidate genes of Thrombosis, 16 were found congregated in module#3, making little over 50% of total gene population of the module containing 30 edges with total of 172 inter molecular interactions. Interestingly, in module#3 most proteins, that are part of the SARS-CoV-2/human interactome are nuclear transporter in their core molecular functionality and known to physically interact with NSP9 and Orf6 gene of SARS-CoV-2. Exclusive, close network of module#3 partner molecules require addition of one more candidate gene namely, NUP85 that may form the molecular basis of COVID-19 associated thrombosis (Figure 1). Biological functions and KEGG analysis of the holistic network and modular compartment further strengthen the predicted candidate gene status as central to the disease biology. Conclusion: The candidate genes identified in the study could later be used as markers for prognosis of the pathology of the COVID-19 for thrombosis and/or developing therapeutic intervention. Additionally, this approach may be helpful in differentiating COVID-19 severity and persistent pathogenesis observed in long COVID syndrome. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

To Improve Prediction of Binding Residues With DNA, RNA, Carbohydrate, and Peptide Via Multi-Task Deep Neural Networks.

The interactions of proteins with DNA, RNA, peptide, and carbohydrate play key roles in various biological processes. The studies of uncharacterized protein-molecules interactions could be aided by accurate predictions of residues that bind with partner molecules. However, the existing methods for predicting binding residues on proteins remain of relatively low accuracies due to the limited number of complex structures in databases. As different types of molecules partially share chemical mechanisms, the predictions for each molecular type should benefit from the binding information with other molecule types. In this study, we employed a multiple task deep learning strategy to develop a new sequence-based method for simultaneously predicting binding residues/sites with multiple important molecule types named MTDsite. By combining four training sets for DNA, RNA, peptide, and carbohydrate-binding proteins, our method yielded accurate and robust predictions with AUC values of 0.852, 0836, 0.758, and 0.776 on their respective independent test sets, which are 0.52 to 6.6% better than other state-of-the-art methods. To my best knowledge, this is the first method using multi-task framework to predict multiple molecular binding sites simultaneously.