Deepan S. H. Shah

Neural differentiation regulated by biomimetic surfaces presenting motifs of extracellular matrix proteins

The interaction between cells and the extracellular matrix (ECM) is essential during development. To elucidate the function of ECM proteins on cell differentiation, we developed biomimetic surfaces that display specific ECM peptide motifs in a controlled manner. Presentation of ECM domains for collagen, fibronectin, and laminin influenced the formation of neurites by differentiating PC12 cells. The effect of these peptide sequences was also tested on the development of adult neural stem/progenitor cells. In this system, collagen I and fibronectin induced the formation of beta-III-tubulin positive cells, whereas collagen IV reduced such differentiation. Biomimetic surfaces composed of multiple peptide types enabled the combinatorial effects of various ECM motifs to be studied. Surfaces displaying combined motifs were often predictable as a result of the synergistic effects of ECM peptides studied in isolation. For example, the additive effects of fibronectin and laminin resulted in greater expression of beta-III-tubulin positive cells, whereas the negative effect of the collagen IV domain was canceled out by coexpression of collagen I. However, simultaneous expression of certain ECM domains was less predictable. These data highlight the complexity of the cellular response to combined ECM signals and the need to study the function of ECM domains individually and in combination.

Monitoring the assembly of antibody-binding membrane protein arrays using polarised neutron reflection

Protein arrays are used in a wide range of applications. The array described here binds IgG antibodies, produced in rabbit, to gold surfaces via a scaffold protein. The scaffold protein is a fusion of the monomeric E. coli porin outer membrane protein A (OmpA) and the Z domain of Staphylococcus aureus protein A. The OmpA binds to gold surfaces via a cysteine residue in a periplasmic turn and the Z domain binds immunoglobulins via their constant region. Polarised Neutron Reflection is used to probe the structure perpendicular to the gold surface at each stage of the assembly of the arrays. Polarised neutrons are used as this provides a means of achieving extra contrast in samples having a magnetic metal layer under the gold surface. This contrast is attained without resorting to hydrogen/deuterium exchange in the biological layer. Polarised Neutron Reflection allows for the modelling of many and complex layers with good fits. The total thickness of the biological layer immobilised on the gold surface is found to be 187 Å and the layer can thus far be separated into its lipid, protein and solvent parts.

Self-assembling layers created by membrane proteins on gold

Membrane systems are based on several types of organization. First, amphiphilic lipids are able to create monolayer and bilayer structures which may be flat, vesicular or micellar. Into these structures membrane proteins can be inserted which use the membrane to provide signals for lateral and orientational organization. Furthermore, the proteins are the product of highly specific self-assembly otherwise known as folding, which mostly places individual atoms at precise places in three dimensions. These structures all have dimensions in the nanoscale, except for the size of membrane planes which may extend for millimetres in large liposomes or centimetres on planar surfaces such as monolayers at the air/water interface. Membrane systems can be assembled on to surfaces to create supported bilayers and these have uses in biosensors and in electrical measurements using modified ion channels. The supported systems also allow for measurements using spectroscopy, surface plasmon resonance and atomic force microscopy. By combining the roles of lipids and proteins, highly ordered and specific structures can be self-assembled in aqueous solution at the nanoscale.

Self-Assembly of Protein Monolayers Engineered for Improved Monoclonal Immunoglobulin G Binding

Bacterial outer membrane proteins, along with a filling lipid molecule can be modified to form stable self-assembled monolayers on gold. The transmembrane domain of Escherichia coli outer membrane protein A has been engineered to create a scaffold protein to which functional motifs can be fused. In earlier work we described the assembly and structure of an antibody-binding array where the Z domain of Staphylococcus aureus protein A was fused to the scaffold protein. Whilst the binding of rabbit polyclonal immunoglobulin G (IgG) to the array is very strong, mouse monoclonal IgG dissociates from the array easily. This is a problem since many immunodiagnostic tests rely upon the use of mouse monoclonal antibodies. Here we describe a strategy to develop an antibody-binding array that will bind mouse monoclonal IgG with lowered dissociation from the array. A novel protein consisting of the scaffold protein fused to two pairs of Z domains separated by a long flexible linker was manufactured. Using surface plasmon resonance the self-assembly of the new protein on gold and the improved binding of mouse monoclonal IgG were demonstrated.

Reversible Non‐Stick Behaviour of a Bacterial Protein Polymer Provides a Tuneable Molecular Mimic for Cell and Tissue Engineering

Yersina pestis, the bubonic plague bacterium, is coated with a polymeric protein hydrogel for protection from host defences. The protein, which is robust and non-stick, resembles structures found in many eukaryotic extracellular-matrix proteins. Cells grown on the natural polymer cannot adhere and grow poorly; however, when cell-adhesion motifs are inserted into the protein, the cells proliferate.

Modular Protein Engineering Approach to the Functionalization of Gold Nanoparticles for Use in Clinical Diagnostics

Functional protein–gold nanoparticle (AuNP) conjugates have a wide variety of applications including biosensing and drug delivery. Correct protein orientation, which is important to maintain functionality on the nanoparticle surface, can be difficult to achieve in practice, and dedicated protein scaffolds have been used on planar gold surfaces to drive the self-assembly of oriented protein arrays. Here we use the transmembrane domain of Escherichia coli outer membrane protein A (OmpATM) to create protein–AuNP conjugates. The addition of a single cysteine residue into a periplasmic loop, to create cysOmpATM, drives oriented assembly and increased equilibrium binding. As the protein surface concentration increases, the sulfur–gold bond in cysOmpATM creates a more densely populated AuNP surface than the poorly organized wtOmpATM layer. The functionalization of AuNP improved both their stability and homogeneity. This was further exploited using multidomain protein chimeras, based on cysOmpATM, which were shown to form ordered protein arrays with their functional domains displayed away from the AuNP surface. A fusion with protein G was shown to specifically bind antibodies via their Fc region. Next, an in vitro selected single chain antibody (scFv)-cysOmpATM fusion protein, bound to AuNP, detected influenza A nucleoprotein, a widely used antigen in diagnostic assays. Finally, using the same scFv-cysOmpATM–AuNP conjugates, a prototype lateral flow assay for influenza demonstrated the utility of fully recombinant self-assembling sensor layers. By simultaneously removing the need for both animal antibodies and a separate immobilization procedure, this technology could greatly simplify the development of a range of in vitro diagnostics.

Presentation of extracellular matrix motifs by biomimetic substrates to control cellular attachment and differentiation

Mutations in the gene encoding a nuclear intermediate filament protein, lamin A, cause a spectra of human age-related diseases and premature ageing syndromes, affecting a number of somatic tissues including muscle, heart, adipose, bone, neurons and skin. One disease mechanism for lamin diseases proposes that lamin A mutations impair the control of adult stem cell proliferation via retinoblastoma protein (Rb) pathway, which has a critical role in the maintenance of mammalian stem cell populations. Recently, we demonstrated the role of lamin A in Rb-dependent cell cycle regulation and maintenance of proliferation in adult skin cells. To further understand the role of lamin A in longevity and maintenance of proliferation we studied its role in ageing of human dermal fibroblasts (HDF) in vitro, which undergo a progressive loss of proliferative cells and an accumulation of irreversibly arrested senescent cells during ageing. Our results show that human fibroblasts aged in vitro acquire a range of aberrant nuclear phenotypes characteristic of progeroid human fibroblasts. Moreover, we show using three different biochemical techniques, including 2D-gel electrophoresis and mass spectrometry, glutathione blot assays and immunoprecipitation methods, that the C-terminal specific cysteine residues in lamin A undergo oxidative modifications (S-glutathiolation and other irreversible oxidative modifications) in senescent fibroblasts. These modifications inhibit the formation of higher-order disulphidelinked forms of lamin A in senescent fibroblasts as shown by cysteine cross-linking assays. In addition, during biochemical fractionation of senescent fibroblasts, these modifications led to a partial proteolysis of lamin A within its C-terminal domain. Consequently, lamin A fails to tether retinoblastoma protein (pRb) within the nuclei of senescent fibroblasts. Consistent with these findings, addition of extracts from senescent fibroblasts to a Xenopus in vitro nuclear assembly system caused oxidative modifications to C-terminal cysteine residues in Xenopus lamin LIII and inhibited nuclear lamina assembly and DNA replication. Our findings suggest that lamin A acts as an oxidative stress sensor and is a central component of senescence signalling. We propose a novel model for ageing of human fibroblasts in vitro whereby the accumulation of oxidative damage to lamin A contributes to senescence signalling by de-stabilising the nuclear architecture. This novel model of ageing by lamin A redox state may explain the impaired maintenance of cells and tissues and decreased longevity in patients with lamin A mutations and may help develop future drug treatments based on anti-oxidant therapy.