Alpha-Synuclein Aggregation Mechanism in the Presence of Nanomaterials.

Parkinson's disease (PD) is characterized by the toxic oligomeric and fibrillar phases formed by monomeric alpha-synuclein (α-syn). Certain nanoparticles have been demonstrated to promote protein aggregation, while other nanomaterials have been found to prevent the process. In the current work, we use nuclear magnetic resonance spectroscopy in conjunction with isothermal titration calorimetry to investigate the cause and mechanism of these opposing effects at the amino acid protein level. The interaction of α-syn with two types of nanomaterials was considered: citrate-capped gold nanoparticles (AuNPs) and graphene oxide (GO). In the presence of AuNPs, α-syn aggregation is accelerated, whereas in the presence of GO, aggregation is prevented. The study indicates that GO sequesters the NAC region of α-syn monomers through electrostatic and hydrophobic interactions, leading to a reduced elongation rate, and AuNPs leave the NAC region exposed while binding the N-terminus, leading to higher aggregation. The protein's inclination toward quicker aggregation is explained by the binding of the N-terminus of α-syn with the gold nanoparticles. Conversely, a comparatively stronger interaction with GO causes the nucleation and growth phases to be postponed and inhibits intermolecular interactions. Our finding offers novel experimental insights at the residue level regarding the aggregation of α-syn in the presence of various nanomaterials and creates new opportunities for the development of suitably functionalized nanomaterial-based therapeutic reagents against Parkinson's and other neurodegenerative diseases.

A One-Dimensional Phase-Modulated (PM) NMR Experiment for Differentiating Spin Systems in Small Molecules Mixtures: With Application to Chiral Discrimination and Bicomponent Organic Systems.

A one-dimensional phase-modulated NMR experiment, which distinguishes the partially resolved peaks and accelerates the data acquisition due to reduced dimensionality, is reported for differentiating spin systems, with application to chiral discrimination. The multifarious utility of the technique is demonstrated in plenteous examples.

IGF-dependent dynamic modulation of a protease cleavage site in the intrinsically disordered linker domain of human IGFBP2.

Functional regulation via conformational dynamics is well known in structured proteins but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy, we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 (hIGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of hIGFBP2, the linker domain (L-hIGFBP2) retains its intrinsic disorder upon binding IGF-1, but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.

Sustained AMPK Activation and Proline Metabolism Play Critical Roles in the Survival of Matrix-Deprived Transformed Cells.

Attachment to the matrix is critical for the survival of adherent cells, whereas detachment triggers death by apoptosis. Therefore, solid tumors must acquire the ability to survive the stress of matrix-detachment to transit through circulation and seed metastases. Although a central role for energy metabolism in cancer progression is well established, what distinguishes its role in the cellular state of the matrix-deprived form compared to the matrix-attached form is not fully understood yet. Using an in vitro transformation model dependent on simian virus 40 (SV40) small t (ST) antigen for cellular survival and proliferation in matrix-deprived conditions, we demonstrate that 5'-adenosine monophosphate-activated protein kinase (AMPK) activity is elevated and sustained under matrix-deprived conditions in ST-expressing fibroblasts. Additionally, these cells display elevated energy (ATP) levels under matrix-deprived conditions in contrast to cells lacking ST expression. The elevated ATP levels are coupled to increased levels of proline in ST-expressing cells, as revealed by metabolomics studies. The AMPK-dependent upregulation of proline oxidase, an enzyme of proline degradation, is a key link for elevated ATP levels. This functional link is further established by proline supplementation concomitant with AMPK activation in matrix-deprived cells lacking ST antigen, yielding ATP and enhancing survival. Thus, our data establishes a key role for AMPK-dependent regulation of proline metabolism in mediating energy homeostasis and promoting survival of matrix-deprived cells. These findings identify key markers that distinguish the metabolic states of matrix-detached and matrix-attached transformed cells and have implications in developing novel therapeutic strategies for specifically targeting matrix-detached metastasizing cancer cells.

Effect of PEGylation on Host Defense Peptide Complexation with Bacterial Lipopolysaccharide.

Conjugation with poly(ethylene glycol) ("PEGylation") is a widely used approach for improving the therapeutic propensities of peptide and protein drugs through prolonging bloodstream circulation, reducing toxicity and immunogenicity, and improving proteolytic stability. In the present study, we investigate how PEGylation affects the interaction of host defense peptides (HDPs) with bacterial lipopolysaccharide (LPS) as well as HDP suppression of LPS-induced cell activation. In particular, we investigate the effects of PEGylation site for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide displaying potent anti-inflammatory effects, primarily provided by its N-terminal part. PEGylation was performed either in the N-terminus, the C-terminus, or in both termini, keeping the total number of ethylene groups (n = 48) constant. Ellipsometry showed KYE28 to exhibit pronounced affinity to both LPS and its hydrophobic lipid A moiety. The PEGylated peptide variants displayed lower, but comparable, affinity for both LPS and lipid A, irrespective of the PEGylation site. Furthermore, both KYE28 and its PEGylated variants triggered LPS aggregate disruption. To investigate the peptide structure in such LPS complexes, a battery of nuclear magnetic resonance (NMR) methods was employed. From this, it was found that KYE28 formed a well-folded structure after LPS binding, stabilized by hydrophobic domains involving aromatic amino acids as well as by electrostatic interactions. In contrast, the PEGylated peptide variants displayed a less well-defined secondary structure, suggesting weaker LPS interactions in line with the ellipsometry findings. Nevertheless, the N-terminal part of KYE28 retained helix formation after PEGylation, irrespective of the conjugation site. For THP1-Xblue-CD14 reporter cells, KYE28 displayed potent suppression of LPS activation at simultaneously low cell toxicity. Interestingly, the PEGylated KYE28 variants displayed similar or improved suppression of LPS-induced cell activation, implying the underlying key role of the largely retained helical structure close to the N-terminus, irrespective of PEGylation site. Taken together, the results show that PEGylation of HDPs can be done insensitively to the conjugation site without losing anti-inflammatory effects, even for peptides inducing such effects through one of its termini.

PD-1 derived CA-170 is an oral immune checkpoint inhibitor that exhibits preclinical anti-tumor efficacy.

Small molecule immune checkpoint inhibitors targeting PD-1 and other pathways may offer advantages including ease of dosing, ability to manage immune-related adverse events (irAEs) due to their shorter pharmacokinetic exposure and opportunity to target more than one pathway for improving efficacy. Here we describe the identification and characterization of CA-170, an amino acid inspired small molecule inhibitor of PD-L1 and VISTA derived from the interface of PD-1 and PD-L1. CA-170 exhibited potent rescue of proliferation and effector functions of T cells inhibited by PD-L1/L2 and VISTA with selectivity over other immune checkpoint proteins as well as a broad panel of receptors and enzymes. Observed blocking of PD-L1 signaling and binding to PD-L1 in the cellular context without preventing the assembly of PD-1:PD-L1 complex support the formation of a defective ternary complex as the mechanism of action of CA-170. Oral administration of CA-170 resulted in increased proliferation and activation of T cells in the tumor, and significant anti-tumor efficacy in a number of immunocompetent mouse tumor models either as a single agent or in combination with approved therapeutics. These results prompted the advancement of CA-170 to human clinical trials.

Insights into the NF-κB-DNA Interaction through NMR Spectroscopy.

Transcription factors bind specifically to their target elements in the genome, eliciting specific gene expression programs. The nuclear factor-κB (NF-κB) system is a family of proteins comprising inducible transcription activators, which play a critical role in inflammation and cancer. The NF-κB members function as dimers with each monomeric unit binding the κB-DNA. Despite the available structures of the various NF-κB dimers in complex with the DNA, the structural features of these dimers in the nucleic acid-free form are not well-characterized. Using solution NMR spectroscopy, we characterize the structural features of 73.1 kDa p50 subunit of the NF-κB homodimer in the DNA-free form and compare it with the κB DNA-bound form of the protein. The study further reveals that in the nucleic acid-free form, the two constituent domains of p50, the N-terminal and the dimerization domains, are structurally independent of each other. However, in a complex with the κB DNA, both the domains of p50 act as a single unit. The study also provides insights into the mechanism of κB DNA recognition by the p50 subunit of NF-κB.

Domain Stability Regulated through the Dimer Interface Controls the Formation Kinetics of a Specific NF-κB Dimer.

The NF-κB family of transcription factors is a key regulator of the immune response in the vertebrates. The family comprises five proteins that function as dimers formed in various combinations among the members, with the RelA-p50 dimer being physiologically the most abundant. While most of the 15 possible dimers are scarcely present in the cell with some remaining experimentally undetected to date, there are specific gene sets that are only activated by certain sparsely populated NF-κB dimers. The mechanism of transcription activation of such specific genes that are activated only by specific NF-κB dimers remains unclear. Here we show that the dimer interfacial residues control the stabilization of the global hydrogen bond network of the NF-κB dimerization domain, which, in turn, controls the thermodynamic stabilization of different NF-κB dimers. The relatively low thermodynamic stability of the RelA-RelA homodimer is critical as it facilitates the formation of the more stable RelA-p50 heterodimer. Through the modulation of the thermodynamic stability of the RelA-RelA homodimer, the kinetics of the RelA-p50 heterodimer formation can be regulated. This phenomenon provides an insight into the mechanism of RelA-RelA specific target gene regulation in physiology.

NMR-based metabolomics with enhanced sensitivity.

NMR-based metabolomics, which emerged along with mass spectrometry techniques, is the preferred method for studying metabolites in medical research and food industries. However, NMR techniques suffer from inherently low sensitivity, regardless of their superior reproducibility. To overcome this, we made two beneficial modifications: we detuned the probe to reach a position called "Spin Noise Tuning Optimum" (SNTO), and we replaced the conventional cylindrical 5 mm NMR tube with an electric field component-optimized shaped tube. We found that concerted use of both modifications can increase the sensitivity (signal to noise ratio per unit volume) and detection of metabolites and decrease the measurement time by order of magnitude. In this study, we demonstrate and discuss the achieved signal enhancement of metabolites on model non-human (bovine serum, amino acid standard mixture) and human urine samples.

Rapid nuclear magnetic resonance data acquisition with improved resolution and sensitivity for high-throughput metabolomic analysis.

Nuclear magnetic resonance (NMR)-based metabolomics has witnessed rapid advancements in recent years with the continuous development of new methods to enhance the sensitivity, resolution, and speed of data acquisition. Some of the approaches were earlier used for peptide and protein resonance assignments and have now been adapted to metabolomics. At the same time, new NMR methods involving novel data acquisition techniques, suited particularly for high-throughput analysis in metabolomics, have been developed. In this review, we focus on the different sampling strategies or data acquisition methods that have been developed in our laboratory and other groups to acquire NMR spectra rapidly with high sensitivity and resolution for metabolomics. In particular, we focus on the use of multiple receivers, phase modulation NMR spectroscopy, and fast-pulsing methods for identification and assignments of metabolites.

The utility of nuclear magnetic resonance spectroscopy in assisted reproduction.

Infertility affects approximately 15-20% of individuals of reproductive age worldwide. Over the last 40 years, assisted reproductive technology (ART) has helped millions of childless couples. However, ART is limited by a low success rate and risk of multiple gestations. Devising methods for selecting the best gamete or embryo that increases the ART success rate and prevention of multiple gestation has become one of the key goals in ART today. Special emphasis has been placed on the development of non-invasive approaches, which do not require perturbing the embryonic cells, as the current morphology-based embryo selection approach has shortcomings in predicting the implantation potential of embryos. An observed association between embryo metabolism and viability has prompted researchers to develop metabolomics-based biomarkers. Nuclear magnetic resonance (NMR) spectroscopy provides a non-invasive approach for the metabolic profiling of tissues, gametes and embryos, with the key advantage of having a minimal sample preparation procedure. Using NMR spectroscopy, biologically important molecules can be identified and quantified in intact cells, extracts or secretomes. This, in turn, helps to map out the active metabolic pathways in a system. The present review covers the contribution of NMR spectroscopy in assisted reproduction at various stages of the process.

Influence of Oxidation Degree of Graphene Oxide on Its Nuclear Relaxivity and Contrast in MRI.

Graphene oxide (GO) serves as a versatile platform for various applications, with the oxygen content of GO playing an important role in governing its properties. In the present study, different GO types covering a wide range of oxidation degree were prepared using our newly developed two-step method involving ball milling of graphite followed by its oxidation to GO. In addition to the variations in their physicochemical properties, the different GO types exhibited differences in proton relaxivity due to their paramagnetic nature. Nuclear magnetic resonance spectroscopy studies showed that the degree of oxidation of GO perturbs its nuclear relaxation properties and, together with intercalated Mn2+ ions, provides large contrast variation in magnetic resonance imaging (MRI). The study for the first time reveals that the surface chemistry of GO affects its relaxivity and opens up new avenues for developing tunable GO-based contrast agents in magnetic resonance imaging for diagnostics and therapies.

Hemoglobin Dynamics in Solution vis-à-vis Under Confinement: An Electrochemical Perspective.

Confining heme protein in silico often leads to beneficial functionalities such as an enhanced electrochemical response from the heme center. This can be harnessed to design effective biosensors for medical diagnostics. Proteins under confinement, surface confinement on the electrode to be precise, have more ordered and monodisperse structure compared to the protein in bulk solution. As the electrochemical response of a protein comes from those protein molecules that are confined within the electrical double layer across the electrode-electrolyte interface, it is expected that restriction of conformational fluctuations of the polymeric protein will help in enhancement of the electrochemical response. This is probably the prima facie reason for electrochemical response enhancement under confinement. We examine the dynamic features of hemoglobin under confinement vis-à-vis that in bulk solution. We use a variety of spectroscopic techniques across a wide time-space window to establish the following facts: (a) hardening of the protein polypeptide backbone, (b) slowing down of protein diffusion, (c) increase in relaxation times in NMR, and (d) slowing down of dielectric relaxation times under confinement. This indicates an overall quenching of protein dynamics when the protein is confined inside silica matrix. Thus, we hypothesize that along with retention of secondary structure, this quenching of dynamics contributes to the enhancement of electrochemical response observed.

Modulation of protein-graphene oxide interactions with varying degrees of oxidation.

The degree of oxidation of graphene oxide (GO) has been shown to be important for its toxicity and drug-loading efficiency. However, the effect of its variations on GO-protein interaction remains unclear. Here, we evaluate the effect of the different oxidation degrees of GO on its interaction with human ubiquitin (8.6 kDa) using solution state nuclear magnetic resonance (NMR) spectroscopy in combination with other biophysical techniques. Our findings show that the interaction between the protein and the different GO samples is weak and electrostatic in nature. It involves fast dynamic exchange of the protein molecules from the surface of the GO. As the oxidation degree of the GO increases, the extent of the interaction with the protein changes. The interaction of the protein with GO can thus be modulated by tuning the degree of oxidation. This study opens up new avenues to design appropriate graphenic materials for use in various biomedical fields such as drug delivery, biomedical devices and imaging.

Backbone resonance assignments of the dimeric domain of the p50 NF-kappaB subunit.

The Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) is a family of transcription factor recognizing a 9-11 base pair kappaB sites on the promoter/enhancer region of their target genes. The family comprises of five members forming dimers amongst themselves in various combinations. Here we report the backbone resonance assignments of the 24 kDa homodimer of the p50 subunit of NF-kappaB. This is the first step towards understanding the mechanism of dimer formation in solution. The secondary structure derived from the chemical shifts for the dimer is largely consistent with that observed in the available crystal structures of the protein in DNA-bound form.

Fast NMR Methods for Identification of Resonances and Metabolic Pathways.

High-throughput analysis of NMR data in metabolomics involves both rapid data acquisition and analysis. We describe here a data collection and analysis protocol, which enables fast multidimensional NMR data acquisition and automated analysis of NMR spectra to rapidly identify the metabolites and assign them to active metabolic pathways in the system.

Rapid NMR assignments of intrinsically disordered proteins using two-dimensional 13C-detection based experiments.

An approach for rapid backbone resonance assignments in proteins using only two 2D NMR experiments is presented. The new method involves a combination of high-resolution 13Cα-detected NMR experiments and selective unlabeling of amino acid residues. The 13C detected 2D hNCA and 2D hNcoCA spectra of a uniformly labeled sample of the protein are analysed in concert with the 2D hNCA spectrum obtained for a selectively unlabeled sample. The combinatorial set of amino acid residues for selective unlabeling is chosen optimally to maximize the assignments. The method is useful for rapid assignment of proteins with low stability such as intrinsically disordered proteins and is applicable to deuterated proteins. This approach helped in assignments of 14.5 kDa human α-synuclein during the course of its aggregation.

A Peptide-Nanoparticle System with Improved Efficacy against Multidrug Resistant Bacteria.

The recent rise of multidrug resistant microbial strains requires development of new and novel therapeutic alternatives. In this study, we present a novel antibacterial system that comprises of modified naturally abundant antimicrobial peptides in conjugation with silver nanoparticles. Further, we propose a simple route to incorporate a cysteine residue either at the N- or C-terminal of the parent peptide. Tagging a cysteine residue at the terminals not only enhances the binding propensity of the resultant peptide with the silver nanoparticle, but also increases its antimicrobial property against several pathogenic bacterial strains including K. pneumoniae. The minimum inhibitory concentration (MIC) values of the cysteine tagged nanoconjugates were obtained in the range of 5-15 µM compared to 50 µM for peptides devoid of the cysteines. The origin and mechanism of such improved activity of the conjugates were investigated using NMR spectroscopy and molecular dynamics (MD) simulations. The application of 13C-isotope labelled media to track the metabolic lifecycle of E. coli cells provided further insights into the system. MD simulations showed that pore formation in membrane bilayer is mediated through a hydrophobic collapse mechanism. The design strategy described herein opens up new-avenues for using biocompatible nanomedicines as a potential alternative to conventional antibiotics.

Enhanced stability of an intrinsically disordered protein against proteolytic cleavage through interactions with silver nanoparticles.

Intrinsically disordered proteins (IDPs), being sensitive to proteolytic degradation both in vitro and in vivo, can be stabilized by the interactions with various binding partners. Here, we show for the first time that silver nanoparticles (AgNPs) have the ability to enhance the half-life of an IDP, thereby rendering it stable for a month against proteolytic degradation. The conjugate of the unstructured linker domain of human insulin-like growth factor binding protein-2 (L-hIGFBP2) with 10 nm citrate-capped AgNPs was studied using two-dimensional NMR spectroscopy and other biophysical techniques. Our studies reveal the extent and nature of residue-specific interactions of the IDP with AgNPs. These interactions mask proteolysis-prone sites of the IDP and stabilize it. This study opens new avenues for the design of appropriate nanoparticles targeting IDPs and for storage, stabilization and delivery of IDPs into cells in a stable form.

Sperm-mediated DNA lesions alter metabolite levels in spent embryo culture medium.

Paternal genetic alterations may affect embryo viability and reproductive outcomes. Currently it is unknown whether embryo metabolism is affected by sperm-mediated abnormalities. Hence, using a mouse model, this study investigated the response to paternally transmitted DNA lesions on genetic integrity and metabolism in preimplantation embryos. Spent embryo culture media were analysed for metabolites by nuclear magnetic resonance spectroscopy and embryonic genetic integrity was determined by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay on embryonic Day 4.5 (E4.5). Metabolic signatures were compared between normally derived embryos (control) and embryos derived from spermatozoa carrying induced DNA lesions (SDL). SDL embryos showed a significant reduction in blastocyst formation on E3.5 and E4.5 (P<0.0001) and had an approximately 2-fold increase in TUNEL-positive cells (P<0.01). A cohort of SDL embryos showing delayed development on E4.5 had increased uptake of pyruvate (P<0.05) and released significantly less alanine (P<0.05) to the medium compared with the corresponding control embryos. On the other hand, normally developed SDL embryos had a reduced (P<0.001) pyruvate-to-alanine ratio compared with normally developed embryos from the control group. Hence, the difference in the metabolic behaviour of SDL embryos may be attributed to paternally transmitted DNA lesions in SDL embryos.