Peptide-based allosteric inhibitor targets TNFR1 conformationally active region and disables receptor-ligand signaling complex.

Tumor necrosis factor (TNF) receptor 1 (TNFR1) plays a pivotal role in mediating TNF induced downstream signaling and regulating inflammatory response. Recent studies have suggested that TNFR1 activation involves conformational rearrangements of preligand assembled receptor dimers and targeting receptor conformational dynamics is a viable strategy to modulate TNFR1 signaling. Here, we used a combination of biophysical, biochemical, and cellular assays, as well as molecular dynamics simulation to show that an anti-inflammatory peptide (FKCRRWQWRMKK), which we termed FKC, inhibits TNFR1 activation allosterically by altering the conformational states of the receptor dimer without blocking receptor-ligand interaction or disrupting receptor dimerization. We also demonstrated the efficacy of FKC by showing that the peptide inhibits TNFR1 signaling in HEK293 cells and attenuates inflammation in mice with intraperitoneal TNF injection. Mechanistically, we found that FKC binds to TNFR1 cysteine-rich domains (CRD2/3) and perturbs the conformational dynamics required for receptor activation. Importantly, FKC increases the frequency in the opening of both CRD2/3 and CRD4 in the receptor dimer, as well as induces a conformational opening in the cytosolic regions of the receptor. This results in an inhibitory conformational state that impedes the recruitment of downstream signaling molecules. Together, these data provide evidence on the feasibility of targeting TNFR1 conformationally active region and open new avenues for receptor-specific inhibition of TNFR1 signaling.

Structural Basis of the Binding Mode of the Antineoplastic Compound Motixafortide (BL-8040) in the CXCR4 Chemokine Receptor.

Modulation of the CXCL12-CXCR4 signaling axis is of the utmost importance due to its central involvement in several pathological disorders, including inflammatory diseases and cancer. Among the different currently available drugs that inhibit CXCR4 activation, motixafortide-a best-in-class antagonist of this GPCR receptor-has exhibited promising results in preclinical studies of pancreatic, breast, and lung cancers. However, detailed information on the interaction mechanism of motixafortide is still lacking. Here, we characterize the motixafortide/CXCR4 and CXCL12/CXCR4 protein complexes by using computational techniques including unbiased all-atom molecular dynamics simulations. Our microsecond-long simulations of the protein systems indicate that the agonist triggers changes associated with active-like GPCR conformations, while the antagonist favors inactive conformations of CXCR4. Detailed ligand-protein analysis indicates the importance of motixafortide's six cationic residues, all of which established charge-charge interactions with acidic CXCR4 residues. Furthermore, two synthetic bulky chemical moieties of motixafortide work in tandem to restrict the conformations of important residues associated with CXCR4 activation. Our results not only elucidate the molecular mechanism by which motixafortide interacts with the CXCR4 receptor and stabilizes its inactive states, but also provide essential information to rationally design CXCR4 inhibitors that preserve the outstanding pharmacological features of motixafortide.

Identification of Molecular Determinants in iRhoms1 and 2 That Contribute to the Substrate Selectivity of Stimulated ADAM17.

The metalloprotease ADAM17 is a key regulator of the TNFα, IL-6R and EGFR signaling pathways. The maturation and function of ADAM17 is controlled by the seven-membrane-spanning proteins iRhoms1 and 2. The functional properties of the ADAM17/iRhom1 and ADAM17/iRhom2 complexes differ, in that stimulated shedding of most ADAM17 substrates tested to date can be supported by iRhom2, whereas iRhom1 can only support stimulated shedding of very few ADAM17 substrates, such as TGFα. The first transmembrane domain (TMD1) of iRhom2 and the sole TMD of ADAM17 are important for the stimulated shedding of ADAM17 substrates by iRhom2. However, little is currently known about how the iRhoms interact with different substrates to control their stimulated shedding by ADAM17. To provide new insights into this topic, we tested how various chimeras between iRhom1 and iRhom2 affect the stimulated processing of the EGFR-ligands TGFα (iRhom1- or 2-dependent) and EREG (iRhom2-selective) by ADAM17. This uncovered an important role for the TMD7 of the iRhoms in determining their substrate selectivity. Computational methods utilized to characterize the iRhom1/2/substrate interactions suggest that the substrate selectivity is determined, at least in part, by a distinct accessibility of the substrate cleavage site to stimulated ADAM17. These studies not only provide new insights into why the substrate selectivity of stimulated iRhom2/ADAM17 differs from that of iRhom1/ADAM17, but also suggest new approaches for targeting the release of specific ADAM17 substrates.

Intrapallidal injection of cannabidiol or a selective GPR55 antagonist decreases motor asymmetry and improves fine motor skills in hemiparkinsonian rats.

Cannabidiol (CBD) presents antiparkinsonian properties and neuromodulatory effects, possibly due to the pleiotropic activity caused at multiple molecular targets. Recently, the GPR55 receptor has emerged as a molecular target of CBD. Interestingly, GPR55 mRNA is expressed in the external globus pallidus (GPe) and striatum, hence, it has been suggested that its activity is linked to motor dysfunction in Parkinson's disease (PD). The present study aimed to evaluate the effect of the intrapallidal injection of both CBD and a selective GPR55 antagonist (CID16020046) on motor asymmetry, fine motor skills, and GAD-67 expression in hemiparkinsonian rats. The hemiparkinsonian animal model applied involved the induction of a lesion in male Wistar rats via the infusion of the neurotoxin 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle via stereotaxic surgery. After a period of twenty days, a second surgical procedure was performed to implant a guide cannula into the GPe. Seven days later, lysophosphatidylinositol (LPI), CBD, or CID16020046 were injected once a day for three consecutive days (from the 28th to the 30th day post-lesion). Amphetamine-induced turning behavior was evaluated on the 14th and 30th days post-injury. The staircase test and fine motor skills were evaluated as follows: the rats were subject to a ten-day training period prior to the 6-OHDA injury; from the 15th to the 19th days post-lesion, the motor skills alterations were evaluated under basal conditions; and, from the 28th to the 30th day post-lesion, the pharmacological effects of the drugs administered were evaluated. The results obtained show that the administration of LPI or CBD generated lower levels of motor asymmetry in the turning behavior of hemiparkinsonian rats. It was also found that the injection of CBD or CID16020046, but not LPI, in the hemiparkinsonian rats generated significantly superior performance in the staircase test, in terms of the use of the forelimb contralateral to the 6-OHDA-induced lesion, when evaluated from the 28th to the 30th day post-lesion. Similar results were also observed for superior fine motor skills performance for pronation, grasp, and supination. Finally, the immunoreactivity levels were found to decrease for the GAD-67 enzyme in the striatum and the ipsilateral GPe of the rats injected with CBD and CID16020046, in contrast with those lesioned with 6-OHDA. The results obtained suggest that the inhibitory effects of CBD and CID16020046 on GPR55 in the GPe could be related to GABAergic overactivation in hemiparkinsonism, thus opening new perspectives to explain, at a cellular level, the reversal of the motor impairment observed in PD models.

Possible Co-Evolution of Polyglutamine and Polyproline in Huntingtin Protein: Proline-Rich Domain as Transient Folding Chaperone.

Huntington's disease is an inherited neurodegenerative disorder caused by the overduplication of CAG repeats in the Huntingtin gene. Recent findings revealed that among the orthologs, the expansion of CAG repeats (polyQ) in the Huntingtin gene occurs in tandem with the duplication of CCG repeats (polyP). However, the molecular mechanism of this possible co-evolution remains unknown. We examined the structures of Huntingtin exon 1 (HttEx1) from six species along with five designed mutants. We found that the polyP segments "chaperone" the rest of the HttEx1 by forming ad hoc polyP binding grooves. Such a process elongates the otherwise poorly solvated polyQ domain, while modulating its secondary structure propensity from ß-strands to α-helices. This chaperoning effect is achieved mostly through transient hydrogen bond interactions between polyP and the rest of HttEx1, resulting in a striking golden ratio of ∼2:1 between the chain lengths of polyQ and polyP.

Novel variants of engineered water soluble mu opioid receptors with extensive mutations and removal of cysteines.

We have shown that water-soluble variants of the human mu opioid receptor (wsMOR) containing a reduced number of hydrophobic residues at the lipid-facing residues of the transmembrane (TM) helices can be expressed in E. coli. In this study, we tested the consequences of increasing the number of mutations on the surface of the transmembrane domain on the receptor's aqueous solubility and ligand binding properties, along with mutation of 11 cysteine residues regardless of their solvent exposure value and location in the protein. We computationally engineered 10 different variants of MOR, and tested four of them for expression in E. coli. We found that all four variants were successfully expressed and could be purified in high quantities. The variants have alpha helical structural content similar to that of the native MOR, and they also display binding affinities for the MOR antagonist (naltrexone) similar to the wsMOR variants we engineered previously that contained many fewer mutations. Furthermore, for these full-length variants, the helical content remains unchanged over a wide range of pH values (pH 6 ~ 9). This study demonstrates the flexibility and robustness of the water-soluble MOR variants with respect to additional designed mutations in the TM domain and changes in pH, whereupon the protein's structural integrity and its ligand binding affinity are maintained. These variants of the full-length MOR with less hydrophobic surface residues and less cysteines can be obtained in large amounts from expression in E. coli and can serve as novel tools to investigate structure-function relationships of the receptor.

Substrate-selective protein ectodomain shedding by ADAM17 and iRhom2 depends on their juxtamembrane and transmembrane domains.

The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) regulates EGF-receptor and TNFα signaling, thereby not only protecting the skin and intestinal barrier, but also contributing to autoimmunity. ADAM17 can be rapidly activated by many stimuli through its transmembrane domain (TMD), with the seven membrane-spanning inactive Rhomboids (iRhom) 1 and 2 implicated as candidate regulatory partners. However, several alternative models of ADAM17 regulation exist that do not involve the iRhoms, such as regulation through disulfide bond exchange or through interaction with charged phospholipids. Here, we report that a non-activatable mutant of ADAM17 with the TMD of betacellulin (BTC) can be rescued by restoring residues from the ADAM17 TMD, but only in Adam17-/- cells, which contain iRhoms, not in iRhom1/2-/- cells. We also provide the first evidence that the extracellular juxtamembrane domains (JMDs) of ADAM17 and iRhom2 regulate the stimulation and substrate selectivity of ADAM17. Interestingly, a point mutation in the ADAM17 JMD identified in a patient with Tetralogy of Fallot, a serious heart valve defect, affects the substrate selectivity of ADAM17 toward Heparin-binding epidermal growth factor like growth factor (HB-EGF), a crucial regulator of heart valve development in mice. These findings provide new insights into the regulation of ADAM17 through an essential interaction with the TMD1 and JMD1 of iRhom2.

Oxidovanadium(V) complexes as promising anticancer photosensitizers.

Photodynamic therapy (PDT) is an alternative treatment widely used against cancer. PDT requires molecular systems, known as photosensitizers (PS), which not only exhibit strong absorption at a particular wavelength range, but also need to be selectively accumulated inside cancer cells. PS are activated by specific wavelengths that cause tumor cell death by mechanisms related with oxidative stress. In this paper, three oxidovanadium(V) complexes linked to a Schiff base, which exhibit anticancer activity by displaying desirable accumulation inside malignant cells, are studied using Density Functional Theory (DFT) and Time Dependent-DFT (TD-DFT) methodologies to characterize their structural and photophysical properties as possible PS. The maximum absorption of these complexes in aqueous solution was predicted to be approximately 460 nm presenting a ligand-to-metal charge transfer. Additionally, we describe the photodynamic type reaction that these complexes can undergo when considered as PS candidates. Our results suggest that the system, containing triethylammonium as substituent, is the most suitable complex to act both as PS and as a possible therapeutic candidate in PDT.

Modeling and Structural Characterization of the Sweet Taste Receptor Heterodimer.

Sweet taste receptor, a heterodimer belonging to the class C G-protein coupled receptor (GPCR) family and composed of the T1R2 and T1R3 subunits, is responsible for the perception of natural sugars, sweet proteins, various d-amino acids, as well as artificial sweeteners. Despite the critical importance of the sweet receptor not only in mediating gustation but also in its role in the food industry, the architecture of the T1R2-T1R3 complex and the mechanism by which extracellular stimuli induce conformational changes that are propagated to the intracellular milieu, i.e., the signal transduction pathway, remain largely unknown. Here, we constructed and characterized a full-length structural model of the T1R2-T1R3 receptor, including both the transmembrane (TM) and extracellular (EC) domains of the heterodimer, using comparative modeling and extensive all-atom molecular dynamics simulations. Several heterodimer interfaces were first examined for the TM domain, and conformational changes occurring at the intracellular side and associated with the receptor's activation were characterized. From the analysis on the simulated data, putative allosteric binding sites for ligands, ions, and cholesterol were proposed. Also, insights into the protein interface of the TM domain upon activation are provided. The EC domain of the heterodimer, including both the Venus flytrap and cysteine-rich domains, was also investigated. Several important intersubunit interactions located at regions responsible for the receptor's proper function were observed, which resemble those recently identified in other class C GPCR members. Integration of the results from the TM and EC domains facilitates the generation of a full-length T1R2-T1R3 receptor. These findings along with the full-length structural model of the T1R2-T1R3 receptor provide a structural framework that may assist in understanding the mechanistic details associated with the receptor activation process for the sweet T1R2-T1R3 receptor as well as other members of the same family.

Inhibitory mechanism of 17ß-aminoestrogens in the formation of Aß aggregates.

Alzheimer's disease (AD) is a complex neurodegenerative disorder associated with the aggregation of the amyloid-beta peptide (Aß) into large oligomers and fibrils that damage healthy brain cells. The predominant peptide fragments in the plaques are mainly formed by the Aß1-40 and Aß1-42 peptides, albeit the eleven-residue Aß25-35 segment is largely used in biological studies because it retains the neurotoxic properties of the longer Aß peptides. Recent studies indicate that treatment with therapeutic steroid hormones reduces the progress of the disease in AD models. Particularly, treatment with 17ß-aminoestrogens (AEs) has shown a significant alleviation of the AD development by inhibiting oxidative stress and neuronal death. Yet, the mechanism by which the AE molecules exhibit their beneficial effects remains speculative. To shed light into the molecular mechanism of inhibition of the AD development by AEs, we investigated the possibility of direct interaction with the Aß25-35 peptide. First, we calculate various interacting electronic properties of three AE derivatives as follows: prolame, butolame, and pentolame by performing DFT calculations. To account for the polymorphic nature of the Aß aggregates, we considered four different Aß25-35 systems extracted from AD relevant fibril structures. From the calculation of different electron density properties, specific interacting loci were identified that guided the construction and optimization of various complexes. Interestingly, the results suggest a similar inhibitory mechanism based on the direct interaction between the AEs and the M35 residue that seems to be general and independent of the polymorphic properties of the Aß aggregates. Our analysis of the complex formation provides a structural framework for understanding the AE therapeutic properties in the molecular inhibitory mechanism of Aß aggregation.

Vanadium in Biological Action: Chemical, Pharmacological Aspects, and Metabolic Implications in Diabetes Mellitus.

Vanadium compounds have been primarily investigated as potential therapeutic agents for the treatment of various major health issues, including cancer, atherosclerosis, and diabetes. The translation of vanadium-based compounds into clinical trials and ultimately into disease treatments remains hampered by the absence of a basic pharmacological and metabolic comprehension of such compounds. In this review, we examine the development of vanadium-containing compounds in biological systems regarding the role of the physiological environment, dosage, intracellular interactions, metabolic transformations, modulation of signaling pathways, toxicology, and transport and tissue distribution as well as therapeutic implications. From our point of view, the toxicological and pharmacological aspects in animal models and humans are not understood completely, and thus, we introduced them in a physiological environment and dosage context. Different transport proteins in blood plasma and mechanistic transport determinants are discussed. Furthermore, an overview of different vanadium species and the role of physiological factors (i.e., pH, redox conditions, concentration, and so on) are considered. Mechanistic specifications about different signaling pathways are discussed, particularly the phosphatases and kinases that are modulated dynamically by vanadium compounds because until now, the focus only has been on protein tyrosine phosphatase 1B as a vanadium target. Particular emphasis is laid on the therapeutic ability of vanadium-based compounds and their role for the treatment of diabetes mellitus, specifically on that of vanadate- and polioxovanadate-containing compounds. We aim at shedding light on the prevailing gaps between primary scientific data and information from animal models and human studies.

Orientational Binding of DNA Guided by the C2N Template.

A detailed understanding of the interactions between biomolecules and nanomaterial surfaces is critical for the development of biomedical applications of these nanomaterials. Here, we characterized the binding patterns and dynamics of a double stranded DNA (dsDNA) segment on the recently synthesized nitrogenized graphene (C2N) with both theoretical (including classical and quantum calculations) and experimental approaches. Our results show that the dsDNA repeatedly exhibits a strong preference in its binding mode on the C2N substrate, displaying an upright orientation that is independent of its initial configurations. Interestingly, once bound to the C2N monolayer, the transverse mobility of the dsDNA is highly restricted. Further energetic and structural analyses reveal that the strength and position of the binding is guided by the favorable π-π stacking between the dsDNA terminal base pairs and the benzene rings on the C2N surface, accompanied by a simultaneous strong nanoscale dewetting that provides additional driving forces. The periodic atomic charge distributions on C2N (from its unique porous structure) also cause the formation of local highly dense first solvation shell water clusters, which act as further steric hindrance for the dsDNA migration. Furthermore, free energy profiling calculated by the umbrella sampling technique quantitatively supports these observations. When compared to graphene, C2N is found to show a milder attraction to dsDNA, which is confirmed by experiments. This orientational binding of DNA on the C2N substrate might shed light on the design of template-guided nanostructures where their functions can be tuned by specialized biomolecular coating.

Structural modeling defines transmembrane residues in ADAM17 that are crucial for Rhbdf2-ADAM17-dependent proteolysis.

A disintegrin and metalloproteinase 17 (ADAM17) controls the release of the pro-inflammatory cytokine tumor necrosis factor α (TNFα, also known as TNF) and is crucial for protecting the skin and intestinal barrier by proteolytic activation of epidermal growth factor receptor (EGFR) ligands. The seven-membrane-spanning protein called inactive rhomboid 2 (Rhbdf2; also known as iRhom2) is required for ADAM17-dependent TNFα shedding and crosstalk with the EGFR, and a point mutation (known as sinecure, sin) in the first transmembrane domain (TMD) of Rhbdf2 (Rhbdf2sin) blocks TNFα shedding, yet little is known about the underlying mechanism. Here, we used a structure-function analysis informed by structural modeling to evaluate the interaction between the TMD of ADAM17 and the first TMD of Rhbdf2, and the role of this interaction in Rhbdf2-ADAM17-dependent shedding. Moreover, we show that double mutant mice that are homozygous for Rhbdf2sin/sin and lack Rhbdf1 closely resemble Rhbdf1/2-/- double knockout mice, highlighting the severe functional impact of the Rhbdf2sin/sin mutation on ADAM17 during mouse development. Taken together, these findings provide new mechanistic and conceptual insights into the critical role of the TMDs of ADAM17 and Rhbdf2 in the regulation of the ADAM17 and EGFR, and ADAM17 and TNFα signaling pathways.

Phosphorylation regulates the secondary structure and function of dentin phosphoprotein peptides.

Dentin phosphoprotein (DPP) is the most acidic protein in vertebrates and structurally is classified as an intrinsically disordered protein. Functionally, DPP is related to dentin and bone formation, however the specifics of such association remain unknown. Here, we used atomistic molecular dynamics simulations to screen selected binding domains of DPP onto hydroxyapatite (HA), which is one of its important interacting partners. From these results, we selected a functionally relevant peptide, Ace-SSDSSDSSDSSDSSD-NH2 (named P5) and its phosphorylated form (named P5P), for experimental characterization. SAXS experiments indicated that in solution P5 was disordered, possibly in an extended conformation while P5P displayed more compact globular conformations. Circular dichroism and FTIR confirmed that, either in the presence or absence of Ca2+/HA, P5 adopts a random coil structure, whereas its phosphorylated counterpart, P5P, has a more compact arrangement associated with conformations that display ß-sheet and α-helix motifs when bound to HA. In solution, P5 inhibited HA crystal growth, whereas at similar concentrations, P5P stimulated it. These findings suggest that phosphorylation controls the transient formation of secondary and tertiary structure of DPP peptides, and, most likely of DPP itself, which in turn controls HA growth in solution and possibly HA growth in mineralized tissues.

Human µ Opioid Receptor Models with Evaluation of the Accuracy Using the Crystal Structure of the Murine µ Opioid Receptor.

Models of the human µ opioid receptor were constructed using available G-protein-coupled receptor (GPCR) structures using homology (comparative) modeling techniques. The recent publication of a high-resolution crystal structure of a construct based on the murine µ opioid receptor offers a unique opportunity to evaluate the reliability of the homology models and test the relevance of introducing more templates (known structures) to increase the accuracy of the comparative models. In the first model two templates were used: the ß2 adrenergic and bovine rhodopsin receptors. For the second model, four templates were utilized: the ß2 adrenergic, bovine rhodopsin, ß1 adrenergic, and A2A adenosine receptors. Including additional templates improved the accuracy of structural motifs and other features of the model when the same sequence alignment was used. The predicted structures were especially relevant in the case of important receptor regions such as the DRY motif, which has been associated with receptor activation. Additionally, this study showed that receptor sequence similarity is crucial in homology modeling, as indicated in the case of the highly diverse EC2 loop. This study demonstrates the reliability of the homology modeling technique in the case of the µ opioid receptor, a member of the rhodopsin-like family class of GPCRs. The addition of more templates improved the accuracy of the model. The findings regarding the modeling has significant implication to other GPCRs where the crystal structure is still unknown and suggest that homology modeling techniques can provide high quality structural models for interpreting experimental findings and formulating structurally based hypotheses regarding the activity of these important receptors.

Computational protein design to reengineer stromal cell-derived factor-1α generates an effective and translatable angiogenic polypeptide analog.

BACKGROUND: Experimentally, exogenous administration of recombinant stromal cell-derived factor-1α (SDF) enhances neovasculogenesis and cardiac function after myocardial infarction. Smaller analogs of SDF may provide translational advantages including enhanced stability and function, ease of synthesis, lower cost, and potential modulated delivery via engineered biomaterials. In this study, computational protein design was used to create a more efficient evolution of the native SDF protein. METHODS AND RESULTS: Protein structure modeling was used to engineer an SDF polypeptide analog (engineered SDF analog [ESA]) that splices the N-terminus (activation and binding) and C-terminus (extracellular stabilization) with a diproline segment designed to limit the conformational flexibility of the peptide backbone and retain the relative orientation of these segments observed in the native structure of SDF. Endothelial progenitor cells (EPCs) in ESA gradient, assayed by Boyden chamber, showed significantly increased migration compared with both SDF and control gradients. EPC receptor activation was evaluated by quantification of phosphorylated AKT, and cells treated with ESA yielded significantly greater phosphorylated AKT levels than SDF and control cells. Angiogenic growth factor assays revealed a distinct increase in angiopoietin-1 expression in the ESA- and SDF-treated hearts. In addition, CD-1 mice (n=30) underwent ligation of the left anterior descending coronary artery and peri-infarct intramyocardial injection of ESA, SDF-1α, or saline. At 2 weeks, echocardiography demonstrated a significant gain in ejection fraction, cardiac output, stroke volume, and fractional area change in mice treated with ESA compared with controls. CONCLUSIONS: Compared with native SDF, a novel engineered SDF polypeptide analog (ESA) more efficiently induces EPC migration and improves post-myocardial infarction cardiac function and thus offers a more clinically translatable neovasculogenic therapy.