Structural Adaptability Facilitates Histidine Heme Ligation in a Cytochrome P450

Almost all known members of the cytochrome P450 (CYP) superfamily conserve a key cysteine residue that coordinates the heme iron. Although mutation of this residue abolishes monooxygenase activity, recent work has shown that mutation to either serine or histidine unlocks non-natural carbene- and nitrene-transfer activities. Here we present the first crystal structure of a histidine-ligated P450. The T213A/C317H variant of the thermostable CYP119 from Sulfolobus acidocaldarius maintains heme iron coordination through the introduced ligand, an interaction that is accompanied by large changes in the overall protein structure. We also find that the axial cysteine C317 may be substituted with any other amino acid without abrogating folding and heme cofactor incorporation. Several of the axial mutants display unusual spectral features, suggesting that they have active sites with unique steric and electronic properties. These novel, highly stable enzyme active sites will be fruitful starting points for investigations of non-natural P450 catalysis and mechanisms

cAMP Response Element-Binding Protein Controls the Appearance of Neuron-Like Traits in Chorion Mesenchymal Cells

Mesenchymal stromal cells (MSC) from bone marrow have been reported to undergo the initial phases of neural differentiation in response to an increase of intracellular cAMP. We investigated the possibility that a similar effect applies to chorion-derived MSC

Plasma neurofilament heavy chain levels and disease progression in amyotrophic lateral sclerosis: insights from a longitudinal study

Objective To investigate the role of longitudinal plasma neurofilament heavy chain protein (NfH) levels as an indicator of clinical progression and survival in amyotrophic lateral sclerosis (ALS). Methods A cross-sectional study involving 136 clinically heterogeneous patients with ALS and 104 healthy and neurological controls was extended to include a prospective analysis of 74 of these ALS cases, with samplings at approximately 3-month intervals in a follow-up period of up to 3 years. We analysed the correlation between longitudinal NfH-phosphoform levels and disease progression. Temporal patterns of NfH changes were evaluated using multilevel linear regression. Results Baseline plasma NfH levels were higher than controls only in patients with ALS with short disease duration to baseline sampling. Compared with controls, fast-progressing patients with ALS, particularly those with a short diagnostic latency and disease duration, had higher plasma NfH levels at an early stage and lower levels closer to end-stage disease. Lower NfH levels between visits were associated with rapid functional deterioration. We also detected antibodies against NfH, NfH aggregates and NfH cleavage products. Conclusions Disease progression in ALS involves defined trajectories of plasma NfH levels, reflecting speed of neurological decline and survival. Intervisit plasma NfH changes are also indicative of disease progression. This study confirms that longitudinal measurements of NfH plasma levels are more informative than cross-sectional studies, where the time of sampling may represent a bias in the interpretation of the results. Autoantibodies against NfH aggregates and NfH cleavage products may explain the variable expression of plasma NfH with disease progressionThis project was funded by The Motor Neurone Disease Association (Malaspina/Apr13/6097) and Barts and The London Charities (468/1714). LG is the Graham Watts Senior Research Fellow, funded by The Brain Research Trust and the European Community’s Seventh Framework Programme (FP7/2007-2013)

Plasma Neurofilament Heavy Chain Levels Correlate to Markers of Late Stage Disease Progression and Treatment Response in SOD1(G93A) Mice that Model ALS

Background: Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder characterised by progressive degeneration of motor neurons leading to death, typically within 3–5 years of symptom onset. The diagnosis of ALS is largely reliant on clinical assessment and electrophysiological findings. Neither specific investigative tools nor reliable biomarkers are currently available to enable an early diagnosis or monitoring of disease progression, hindering the design of treatment trials. Methodology/Principal Findings: In this study, using the well-established SOD1G93A mouse model of ALS and a new in-house ELISA method, we have validated that plasma neurofilament heavy chain protein (NfH) levels correlate with both functional markers of late stage disease progression and treatment response. We detected a significant increase in plasma levels of phosphorylated NfH during disease progression in SOD1G93A mice from 105 days onwards. Moreover, increased plasma NfH levels correlated with the decline in muscle force, motor unit survival and, more significantly, with the loss of spinal motor neurons in SOD1 mice during this critical period of decline. Importantly, mice treated with the disease modifying compound arimoclomol had lower plasma NfH levels, suggesting plasma NfH levels could be validated as an outcome measure for treatment trials. Conclusions/Significance: These results show that plasma NfH levels closely reflect later stages of disease progression and therapeutic response in the SOD1G93A mouse model of ALS and may potentially be a valuable biomarker of later disease progression in ALS

Tissue-enhanced plasma proteomic analysis for disease stratification in amyotrophic lateral sclerosis

Motor Neurone Disease Association (Malaspina/Apr13/817–791). Wellcome Trust support to a parallel study (Pathfinder Award, grant number 103208)

Development of Single-Walled Carbon Nanotube Protein Optical Nanosensors

Many diseases, such as neurodegenerative disorders, cancer, and autoimmune diseases are caused by or exhibit symptoms of abnormal regulation of signaling proteins. In order to obtain a molecular understanding of diseases, we require tools capable of probing the intricate signaling pathways that govern biological function. Understanding the role these proteins play as they circulate between cells will allow us to examine disease progression from an intercellular point of view. Currently, it is difficult to study these proteins in their natural environment, in vivo, because they are created and function on very broad time and length scales. Existing protein detection methods have several limitations as they are optimized for intracellular imaging, performed in vitro, require lengthy sample handling, function over short time scales, or have molecular recognition elements that are unstable in biological media.This dissertation presents a modular platform to create optical nanosensors that are able to address current limitations in signaling protein detection. Nanosensor elements require optimization of both signal transduction and molecular recognition elements. Single-walled carbon nanotubes (SWCNTs) are nanoparticles that are ideal signal transducers for biological imaging. SWCNTs have optical properties well-suited for biological sensing such as infinite fluorescence lifetime, no blinking, small size, and fluorescence in the near-infrared region of the electromagnetic spectrum that is least attenuated by biological systems. Several SWCNT nanosensors have already been developed for signaling small molecule targets and peptides, but, to date, none have been created for signaling proteins.In order to create a robust imaging platform based on SWCNTs for sensing signaling proteins, it is possible to couple molecular recognition elements to SWCNT signal transducers using dual noncovalent and covalent functionalization strategies. The development of noncovalent molecular recognition elements is explored using peptide mimetic polymers called peptoids. The discovery of a synthetic peptoid binding loop for wheat germ agglutinin protein as a proof-of-principle case study shows the ability to utilize diverse chemical materials to create a fully synthetic binding element for desired protein targets. Additionally, the development of covalently functionalized SWCNTs is explored for the creation of multifunctional optical SWCNT nanosensors. Here covalently functional groups provide functional handles that work synergistically with noncovalent passivation to enable the development of a diverse nanosensor toolbox.The findings presented in this dissertation lay the foundation of valuable techniques and materials to optimize the binding to and study of signaling proteins. Future work to streamline the development of novel nanosensors is discussed, and the framework for the creation of other biological nanomaterial tools through these noncovalent and covalent techniques is also explored

Surface Engineering of Nanoparticles to Create Synthetic Antibodies.

Surface engineering of nanoparticles has recently emerged as a promising technique for synthetic molecular recognition of biological analytes. In particular, the use of synthetic heteropolymers adsorbed onto the surface of a nanoparticle can yield selective detection of a molecular target. Synthetic molecular recognition has unique advantages in leveraging the photostability, versatility, and exceptional chemical stability of nanomaterials. In particular, single-walled carbon nanotubes (SWNT) exhibit a large Stokes shift and near infrared emission for maximum biological sample transparency. Optical biosensors with high signal transduction and molecular specificity can be synthesized with amphiphilic heteropolymers grafted to SWNT, and discovered by high-throughput screening. Herein, we describe the development and the characterization of surface-engineered nanoparticles, or synthetic antibodies, for protein detection

Synthetic probe development for measuring single or few-cell activity and efflux.

Studying the single cell protein secretome offers the opportunity to understand how a phenotypically heterogeneous population of individual cells contribute to ensemble physiology and signaling. Polarized secretion events such as neurotransmitter release and cytokine signaling necessitates spatiotemporal information to elucidate structure-function relationships. Polymer functionalized single-walled carbon nanotube protein sensor arrays allow microscopic imaging of secreted protein footprints and enable the study of the spatiotemporal heterogeneity of protein secretion at the single-cell level. The protocols for carbon nanotube sensor creation, sensor array preparation, and imaging secreted proteins in both prokaryotic and mammalian cells are presented in this chapter. Secreted RAP1 and HIV-1 integrase proteins were used as proof-of-concept examples. Additionally, we discuss potential variety of protein and non-protein analyte effluxes that can be imaged using this platform, as well as current and future perspectives related to sensor development and deployment

Engineering at the nano-bio interface: harnessing the protein corona towards nanoparticle design and function

Unpredictable and uncontrollable protein adsorption on nanoparticles remains a considerable challenge to achieving effective application of nanotechnologies within biological environments. Nevertheless, engineered nanoparticles offer unprecedented functionality and control in probing and altering biological systems. In this review, we highlight recent advances in harnessing the "protein corona" formed on nanoparticles as a handle to tune functional properties of the protein-nanoparticle complex. Towards this end, we first review nanoparticle properties that influence protein adsorption and design strategies to facilitate selective corona formation, with the corresponding characterization techniques. We next focus on literature detailing corona-mediated functionalities, including stealth to avoid recognition and sequestration while in circulation, targeting of predetermined in vivo locations, and controlled activation once localized to the intended biological compartment. We conclude with a discussion of biocompatibility outcomes for these protein-nanoparticle complexes applied in vivo. While formation of the nanoparticle-corona complex may impede our control over its use for the projected nanobiotechnology application, it concurrently presents an opportunity to create improved protein-nanoparticle architectures by exploiting natural or guiding selective protein adsorption to the nanoparticle surface