Recurrent Herpes Simplex Virus Type 1 (HSV-1) Infection Modulates Neuronal Aging Marks in In Vitro and In Vivo Models

Herpes simplex virus 1 (HSV-1) is a widespread neurotropic virus establishing a life-long latent infection in neurons with periodic reactivations. Recent studies linked HSV-1 to neurodegenerative processes related to age-related disorders such as Alzheimer’s disease. Here, we explored whether recurrent HSV-1 infection might accelerate aging in neurons, focusing on peculiar marks of aged cells, such as the increase in histone H4 lysine (K) 16 acetylation (ac) (H4K16ac); the decrease of H3K56ac, and the modified expression of Sin3/HDAC1 and HIRA proteins. By exploiting both in vitro and in vivo models of recurrent HSV-1 infection, we found a significant increase in H4K16ac, Sin3, and HDAC1 levels, suggesting that the neuronal response to virus latency and reactivation includes the upregulation of these aging markers. On the contrary, we found a significant decrease in H3K56ac that was specifically linked to viral reactivation and apparently not related to aging-related markers. A complex modulation of HIRA expression and localization was found in the brain from HSV-1 infected mice suggesting a specific role of this protein in viral latency and reactivation. Overall, our results pointed out novel molecular mechanisms through which recurrent HSV-1 infection may affect neuronal aging, likely contributing to neurodegeneration.     

Discovery of Highly Potent CRBN Ligands and Insight into Their Binding Mode through Molecular Docking and Molecular Dynamics Simulations

Cereblon (CRBN) is a substrate receptor of E3 ubiquitin ligase as well as the target of thalidomide and lenalidomide, plays a vital role in endogenous protein degradation. In this article, two series of compounds with novel structure were designed, synthesized and evaluated against CRBN. YJ1b, designed based on our previous finding, shown strong binding affinity toward CRBN (IC₅₀=0.206 μM) by forming a salt bridge interaction with amino acid residue Glu377 of CRBN, it was 13-fold compared with that of lenalidomide (IC₅₀=2.694 μM) in TR-FRET assay. YJ2c and YJ2h, two analogs of YJ1b, also exhibit high binding affinity toward CRBN (IC₅₀=0.211 μM and IC₅₀=0.282 μM, respectively). While, molecular docking and 100 ns molecular dynamic simulation studies were conducted to insight into the unique binding mode of YJ1b, YJ2c and YJ2e toward CRBN. The new compounds with special binding mode in this article may serve for the further optimization and discovery of novel high potent CRBN ligands.     

A surface plasmon resonance analysis of the interaction between the antibiotic moenomycin A and penicillin-binding protein 1b

The antibiotic moenomycin A inhibits the biosynthesis of peptidoglycan, the main structural polymer of the bacterial cell wall. The inhibition is based on a reversible binding of the antibiotic to one of the substrate binding sites in enzymes such as penicillin-binding protein (PBP) 1b. A novel assay based on surface plasmon resonance (SPR) has been established that can be used to investigate selective binding of the moenomycin sugar moiety and other transglycosylase inhibitors to this enzyme. Suitable ligands were prepared from moenomycin A and coupled to SPR sensor surfaces. Moenomycin analogues with structural variations were used to perform competitive SPR experiments with PBP 1b. The SPR results confirm for the first time that the trisaccharide fragment of moenomycin A (C-E-F-G-H-I) is the minimal structure that possesses all moieties sufficient for biological activity and for affinity towards PBP 1b. The method seems to be appropriate for use in screens for transglycosylase inhibitors that bind to the moenomycin-binding site of the enzyme.     

电沉积石墨烯的非共价功能化用于SPR核酸传感的协同增敏

研究了一种高效的表面等离子共振（SPR）基DNA分析方法,利用非共价功能化的石墨烯纳米片作为基底,并以酶催化诱导的聚合作为质量中继。该策略有多方面的目标：首先,通过原位优化的石墨烯薄膜的电生成,该过程由原子力显微拓扑图表征,来敏化总体的SPR输出;其次,用于调制镍离子螯合的胺基三乙酸支架,其吸附在石墨烯支撑的苝基衍生物表面,上端生物素化捕获探针可以自组装,并保有一定的方向;最后,通过辣根过氧化物酶标记的报告单元,实时地将苯胺添加剂转化为聚苯胺沉淀,以协同实现信号的放大。运用上述配置,获得了一个对特异性DNA靶标精确且可重现传感的平台,检测下限达到飞摩尔水平,从而展现了以二维纳米材料为独特SPR基础设施的有益探索和开发。该“自下而上”的建构、与置顶“自上而下”的重量反应器,极有可能拓展并移植用于蛋白质的定量。     

Affinity adsorption mechanism studies of adsorbents for oligopeptides using model polymer

To understand the principle of designing efficient adsorbents toward specific peptides, adsorption mechanism between oligopeptide VVRGCTWW (VW-8) and alkylamine modified polyacrylamide adsorbents was studied in this article. Linear models of the adsorbents were used to facilitate various measurements except for adsorption experiment. 2D ¹H NMR experiments reveal the main binding site of VW-8 to the adsorbents is C-terminal Trp. When peptide CTWW (CW-4) was introduced as the reference, the same binding site was found. However, in adsorption experiments, a remarkable difference was observed with the adsorption capacity of the two peptides. According to isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) experiments, which are in good agreement with the adsorption results, VW-8 has much higher affinity to the adsorbents than CW-4 and the main driving force in adsorption process is considered to be electrostatic force. However, the unusual affinity adsorption of two oligopeptides on the adsorbents contrasted to the traditional view of electrostatic interaction. With a comprehensive understanding of above experimental results along with computer-aided analysis, we conclude that the difference in conformation of the two peptides has a great impact on the interaction mode and further affects the affinity between the peptides and the adsorbents. This provides valuable information for the further design of synthetic affinity ligand for VW-8.     

Kunitz-Type Peptides from Sea Anemones Protect Neuronal Cells against Parkinson’s Disease Inductors via Inhibition of ROS Production and ATP-Induced P2X7 Receptor Activation

Parkinson’s disease (PD) is a socially significant disease, during the development of which oxidative stress and inflammation play a significant role. Here, we studied the neuroprotective effects of four Kunitz-type peptides from Heteractis crispa and Heteractis magnifica sea anemones against PD inductors. The peptide HCIQ1c9, which was obtained for the first time, inhibited trypsin less than other peptides due to unfavorable interactions of Arg17 with Lys43 in the enzyme. Its activity was reduced by up to 70% over the temperature range of 60–100 °C, while HCIQ2c1, HCIQ4c7, and HMIQ3c1 retained their conformation and stayed active up to 90–100 °C. All studied peptides inhibited paraquat- and rotenone-induced intracellular ROS formation, in particular NO, and scavenged free radicals outside the cells. The peptides did not modulate the TRPV1 channels but they affected the P2X7R, both of which are considered therapeutic targets in Parkinson’s disease. HMIQ3c1 and HCIQ4c7 almost completely inhibited the ATP-induced uptake of YO-PRO-1 dye in Neuro-2a cells through P2X7 ion channels and significantly reduced the stable calcium response in these cells. The complex formation of the peptides with the P2X7R extracellular domain was determined via SPR analysis. Thus, these peptides may be considered promising compounds to protect neuronal cells against PD inductors, which act as ROS production inhibitors and partially act as ATP-induced P2X7R activation inhibitors.     

CK1δ-Derived Peptides as Novel Tools Inhibiting the Interactions between CK1δ and APP695 to Modulate the Pathogenic Metabolism of APP

Alzheimer’s disease (AD) is the major cause of dementia, and affected individuals suffer from severe cognitive, mental, and functional impairment. Histologically, AD brains are basically characterized by the presence of amyloid plaques and neurofibrillary tangles. Previous reports demonstrated that protein kinase CK1δ influences the metabolism of amyloid precursor protein (APP) by inducing the generation of amyloid-β (Aβ), finally contributing to the formation of amyloid plaques and neuronal cell death. We therefore considered CK1δ as a promising therapeutic target and suggested an innovative strategy for the treatment of AD based on peptide therapeutics specifically modulating the interaction between CK1δ and APP. Initially, CK1δ-derived peptides manipulating the interactions between CK1δ and APP695 were identified by interaction and phosphorylation analysis in vitro. Selected peptides subsequently proved their potential to penetrate cells without inducing cytotoxic effects. Finally, for at least two of the tested CK1δ-derived peptides, a reduction in Aβ levels and amyloid plaque formation could be successfully demonstrated in a complex cell culture model for AD. Consequently, the presented results provide new insights into the interactions of CK1δ and APP695 while also serving as a promising starting point for further development of novel and highly innovative pharmacological tools for the treatment of AD.     

Evaluation of Naturally Occurring HIF-1 Inhibitors for Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare and severe progressive disorder characterized by high pulmonary artery pressure. Chronic hypoxia causes a metabolic disorder and the Warburg effect in pulmonary arterial smooth muscle cells (PASMCs). Pyruvate dehydrogenase kinase 1 (PDK1) is a key enzyme in Warburg effect increased by hypoxia-inducible factor (HIF-1). We constructed a cell-based luciferase assay system for HIF-1 inhibitors. Using this system, six HIF-1 inhibitors were identified. Among these inhibitors, the effect of tagitinin C ( 1 ) on PASMC was investigated. Tagitinin C ( 1 ) clearly decreased the amount of HIF-1β and the HIF-1 target PDK1. This result indicates that HIF-1 inhibitors effectively decrease PDK1 activity, which is a cause of the metabolic disorder and Warburg effect observed in PASMCs. Identifying naturally occurring HIF-1 inhibitors could provide novel insights into the development of PAH medications.     

The Cell Adhesion Molecule L1 Interacts with Methyl CpG Binding Protein 2 via Its Intracellular Domain

Cell adhesion molecule L1 regulates multiple cell functions, and L1 deficiency is linked to several neural diseases. Recently, we have identified methyl CpG binding protein 2 (MeCP2) as a potential binding partner of the intracellular L1 domain. By ELISA we show here that L1’s intracellular domain binds directly to MeCP2 via the sequence motif KDET. Proximity ligation assay with cultured cerebellar and cortical neurons suggests a close association between L1 and MeCP2 in nuclei of neurons. Immunoprecipitation using MeCP2 antibodies and nuclear mouse brain extracts indicates that MeCP2 interacts with an L1 fragment of ~55 kDa (L1−55). Proximity ligation assay indicates that metalloproteases, β-site of amyloid precursor protein cleaving enzyme (BACE1) and ɣ-secretase, are involved in the generation of L1−55. Reduction in MeCP2 expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons as well as the migration of L1-expressing HEK293 cells. Moreover, L1 siRNA, MeCP2 siRNA, or a cell-penetrating KDET-containing L1 peptide leads to reduced levels of myocyte enhancer factor 2C (Mef2c) mRNA and protein in cortical neurons, suggesting that the MeCP2/L1 interaction regulates Mef2c expression. Altogether, the present findings indicate that the interaction of the novel fragment L1−55 with MeCP2 affects L1-dependent functions, such as neurite outgrowth and neuronal migration.     

Cyclic Hexapeptide Mimics of the LEDGF Integrase Recognition Loop in Complex with HIV‐1 Integrase

The p75 splice variant of lens epithelium‐derived growth factor (LEDGF) is a 75 kDa protein, which is recruited by the human immunodeficiency virus (HIV) to tether the pre‐integration complex to the host chromatin and promote integration of proviral DNA into the host genome. We designed a series of small cyclic peptides that are structural mimics of the LEDGF binding domain, which interact with integrase as potential binding inhibitors. Herein we present the X‐ray crystal structures, NMR studies, SPR analysis, and conformational studies of four cyclic peptides bound to the HIV‐1 integrase core domain. Although the X‐ray studies show that the peptides closely mimic the LEDGF binding loop, the measured affinities of the peptides are in the low millimolar range. Computational analysis using conformational searching and free energy calculations suggest that the low affinity of the peptides is due to mismatch between the low‐energy solution and bound conformations.     

Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C-Met by Proteolytic Affinity-Mass Spectrometry

C-Met protein is a glycosylated receptor tyrosine kinase of the hepatocyte growth factor (HGF), composed of an α and a β chain. Upon ligand binding, C-Met transmits intracellular signals by a unique multi-substrate docking site. C-Met can be aberrantly activated leading to tumorigenesis and other diseases, and has been recognized as a biomarker in cancer diagnosis. C-Met aptamers have been recently considered a useful tool for detection of cancer biomarkers. Herein we report a molecular interaction study of human C-Met expressed in kidney cells with two DNA aptamers of 60 and 64 bases (CLN0003 and CLN0004), obtained using the SELEX ( S ystematic E volution of L igands by Ex ponential Enrichment) procedure. Epitope peptides of aptamer-C-Met complexes were identified by proteolytic affinity-mass spectrometry in combination with SPR biosensor analysis (PROTEX-SPR-MS), using high-pressure proteolysis for efficient digestion. High affinities (K_D, 80–510 nM) were determined for aptamer-C-Met complexes, with two-step binding suggested by kinetic analysis. A linear epitope, C-Met (381–393) was identified for CLN0004, while the CLN0003 aptamer revealed an assembled epitope comprised of two peptide sequences, C-Met (524–543) and C-Met (557–568). Structure modeling of C-Met-aptamers were consistent with the identified epitopes. Specificities and affinities were ascertained by SPR analysis of the synthetic epitope peptides. The high affinities of aptamers to C-Met, and the specific epitopes revealed render them of high interest for cellular diagnostic studies.     

Prodynorphin and Proenkephalin in Cerebrospinal Fluid of Sporadic Creutzfeldt–Jakob Disease

Proenkephalin (PENK) and prodynorphin (PDYN) are endogenous opioid peptides mainly produced in the striatum and, to a lesser extent, in the cerebral cortex. Dysregulated metabolism and altered cerebrospinal fluid (CSF) levels of PENK and PDYN have been described in several neurodegenerative diseases. However, no study to date investigated these peptides in the CSF of sporadic Creutzfeldt–Jakob disease (sCJD). Using liquid chromatography-multiple reaction monitoring mass spectrometry, we evaluated the CSF PDYN- and PENK-derived peptide levels in 25 controls and 63 patients with sCJD belonging to the most prevalent molecular subtypes (MM(V)1, VV2 and MV2K). One of the PENK-derived peptides was significantly decreased in each sCJD subtype compared to the controls without a difference among subtypes. Conversely, PDYN-derived peptides were selectively decreased in the CSF of sCJD MV2K, a subtype with a more widespread overall pathology compared to the sCJD MM(V)1 and the VV2 subtypes, which we confirmed by semiquantitative analysis of cortical and striatal neuronal loss and astrocytosis. In sCJD CSF PENK and PDYN were associated with CSF biomarkers of neurodegeneration but not with clinical variables and showed a poor diagnostic performance. CSF PDYN and PENK-derived peptides had no significant diagnostic and prognostic values in sCJD; however, the distinct marker levels between molecular subtypes might help to better understand the basis of phenotypic heterogeneity determined by divergent neuronal targeting.     

The Yersinia adhesin YadA binds to a collagenous triple-helical conformation but without sequence specificity

The Yersinia adhesin A (YadA) is a collagen-binding trimeric autotransporter of Yersinia enterocolitica, an enteropathogen that causes a range of gastroenteric and systemic diseases, and YadA is essential for Y. enterocolitica virulence. Although previous studies suggest a specific binding site in collagen for YadA, we found that recombinant YadA binds to both major cyanogen bromide fragments of collagen type II and the collagen-like model peptide (Pro-Hyp-Gly)(10) [(POG)(10)]. To further characterise the YadA-collagen interaction, we investigated the binding of YadA to (POG)(10) and three other model peptides, (Pro-Pro-Gly)(10) which lacks the hydroxyl groups of (POG)(10), T3-785 which contains a stretch of the collagen type III sequence and Gly(-) which is similar to (POG)(10) but lacks the central glycine. All the peptides except Gly(-) adopt a collagen-like triple-helical conformation at room temperature. All three triple-helical peptides bound to YadA, with (POG)(10) being the tightest, whereas binding of Gly(-) was hardly detectable. The affinity of (POG)(10) for YadA was 0.28 microM by isothermal titration calorimetry and 0.17 microM by surface plasmon resonance (SPR), similar to that of collagen type I. Our results show that a collagen-like triple-helical conformation, strengthened by the presence of hydroxyproline residues, is both necessary and sufficient for YadA binding.     

Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

Peptides constructed from α‐helical subunits of the Lac repressor protein (LacI) were designed then tailored to achieve particular binding kinetics and dissociation constants for plasmid DNA purification and detection. Surface plasmon resonance was employed for quantification and characterization of the binding of double stranded Escherichia coli plasmid DNA (pUC19) via the lac operon (lacO) to “biomimics” of the DNA binding domain of LacI. Equilibrium dissociation constants (K_D), association (k_a), and dissociation rates (k_d) for the interaction between a suite of peptide sequences and pUC19 were determined. K_D values measured for the binding of pUC19 to the 47mer, 27mer, 16mer, and 14mer peptides were 8.8 ± 1.3 × 10^?10 M, 7.2 ± 0.6 × 10^?10 M, 4.5 ± 0.5 × 10^?8 M, and 6.2 ± 0.9 × 10^?6 M, respectively. These findings show that affinity peptides, composed of subunits from a naturally occurring operon–repressor interaction, can be designed to achieve binding characteristics suitable for affinity chromatography and biosensor devices. © 2008 American Institute of Chemical Engineers AIChE J, 2009     

Potent inhibitors of LXXLL-based protein-protein interactions

Protein-protein interactions between estrogen receptors, ERalpha and ERbeta, and their coactivators (CoAs) are an attractive target for drug intervention. This interaction is mediated by a small pentapeptide motif (LXXLL), termed the NR box. Based on this motif, a variety of cyclic and linear peptides were synthesized in order to gain a better understanding of the association of CoA proteins with the ER isoforms. Utilizing a time-resolved florescence-based coactivator interaction assay, we determined the abilities of these peptides to inhibit this interaction. Using molecular modeling and CD spectroscopy, we have examined the structural basis of their bioactivities with both hormone receptor isoforms. Either homocysteine or penicillamine was utilized as a substitute for cysteine in the disulfide-bridged peptides, while tertiary leucine and neopentyl glycine were used as the surrogates for the NR box leucines. The most potent disufide-bridged peptide (K(i)= 70 pM, with ERalpha) incorporates neopentyl glycine in the NR box, while the most active peptide in this series with ERbeta (K(i)=350 pM) incorporates tertiary leucine. Surprisingly, several linear peptides containing a single cysteine residue showed activities with low nanomolar K(i) values. Collectively, our results suggest a synthetic approach for designing potent and selective peptidomimetics for ERalpha and ERbeta interactions with CoA proteins effecting estrogen action.     

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (K_D) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.     

Initial Molecular Recognition Steps of McjA Precursor during Microcin J25 Lasso Peptide Maturation

Microcin J25 (MccJ25) has emerged as an excellent model to understand the maturation of ribosomal precursor peptides into the entangled lasso fold. MccJ25 biosynthesis relies on the post‐translational modification of the precursor McjA by the ATP‐dependent protease McjB and the lactam synthetase McjC. Here, using NMR spectroscopy, we showed that McjA is an intrinsically disordered protein without detectable conformational preference, which emphasizes the active role of the maturation machinery on the three‐dimensional folding of MccJ25. We further showed that the N‐terminal region of the leader peptide is involved in interaction with both maturation enzymes and identified a predominant interaction of V43–S55 in the core McjA sequence with McjC. Moreover, we demonstrated that residues K23–Q34 in the N‐terminal McjA leader peptide tend to adopt a helical conformation in the presence of membrane mimics, implying a role in directing McjA to the membrane in the vicinity of the lasso synthetase/export machinery. These data provide valuable insights into the initial molecular recognition steps in the MccJ25 maturation process.