Using Mutagenesis and Structural Biology to Map the Binding Site for the Plasmodium falciparum Merozoite Protein PfRh4 on the Human Immune Adherence Receptor

To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1–3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1–3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an ∼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.     

Multimeric Growth Hormone Receptor Complexes Serve as Signaling Platforms

Growth hormone (GH) signaling is required for promoting longitudinal body growth, stem cell activation, differentiation, and survival and for regulation of metabolism. Failure to adequately regulate GH signaling leads to disease: excessive GH signaling has been connected to cancer, and GH insensitivity has been reported in cachexia patients. Since its discovery in 1989, the receptor has served a pivotal role as the prototype cytokine receptor both structurally and functionally. Phosphorylation and ubiquitylation regulate the GH receptor (GHR) at the cell surface: two ubiquitin ligases (SCF^βTrCP2 and CHIP) determine the GH responsiveness of cells by controlling its endocytosis, whereas JAK2 initiates the JAK/STAT pathway. We used blue native electrophoresis to identify phosphorylated and ubiquitylated receptor intermediates. We show that GHRs occur as ∼500-kDa complexes that dimerize into active ∼900-kDa complexes upon GH binding. The dimerized complexes act as platforms for transient interaction with JAK2 and ubiquitin ligases. If GH and receptors are made in the same cell (autocrine mode), only limited numbers of ∼900-kDa complexes are formed. The experiments reveal the dynamic changes in post-translational modifications during GH-induced signaling events and show that relatively simple cytokine receptors like GHRs are able to form higher order protein complexes. Insight in the complex formation of cytokine receptors is crucially important for engineering cytokines that control ligand-induced cell responses and for generating a new class of therapeutic agents for a wide range of diseases.     

Type V secretion: From biogenesis to biotechnology

The two membranes of Gram-negative bacteria contain protein machines that have a general function in their assembly. To interact with the extra-cellular milieu, Gram-negatives target proteins to their cell surface and beyond. Many specialized secretion systems have evolved with dedicated translocation machines that either span the entire cell envelope or localize to the outer membrane. The latter act in concert with inner-membrane transport systems (i.e. Sec or Tat). Secretion via the Type V secretion system follows a two-step mechanism that appears relatively simple. Proteins secreted via this pathway are important for the Gram-negative life-style, either as virulence factors for pathogens or by contributing to the survival of non-invasive environmental species. Furthermore, this system appears well suited for the secretion of biotechnologically relevant proteins. In this review we focus on the biogenesis and application of two Type V subtypes, the autotransporters and two-partner secretion (TPS) systems. For translocation across the outer membrane the autotransporters require the assistance of the Bam complex that also plays a generic role in the assembly of outer membrane proteins. The TPS systems do use a dedicated translocator, but this protein shows resemblance to BamA, the major component of the Bam complex. Interestingly, both the mechanistic and more applied studies on these systems have provided a better understanding of the secretion mechanism and the biogenesis of outer membrane proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Monitoring changes in skin temperature associated with exercise in horses on a water treadmill by use of infrared thermography

Infrared thermography (IRT) was used to assess surface temperature change as an indirect measure of muscle activity and exercise associated changes in blood flow in the working hind limb muscles of horses (n=7) undergoing water treadmill exercise. Three treatments were investigated including the treadmill ran dry (TD), water at the height of the proximal interphalangeal joint (PIP) and water at the height of the carpus (CP). Maximum skin surface temperature was recorded from the region of the semitendinosus muscle during exercise at each water height. There was a significant difference in surface hind limb temperature between exercise on the water treadmill ran dry and with water at the height of the PIP and CP (P<0.0001) with hotter temperatures recorded during the TD treatment. There was a greater increase in surface temperature of the hind limbs from pre exercise to maximum temperature during the PIP and CP treatments when compared to the TD treatment, however, this was not significant (P=0.58). There was no significant difference in surface hind limb temperature found between exercise in water at the height of the PIP and water at the height of the CP. The findings from this study suggest that IRT is able to non-invasively detect muscle activity and associated changes in blood flow whilst horses are exercised on a water treadmill. IRT could potentially be used as an alternative method to assess muscle activity and temperature change in an aquatic environment where existing methods present methodological challenges.     

Biomaterials surfaces capable of resisting fungal attachment and biofilm formation

Microbial attachment onto biomedical devices and implants leads to biofilm formation and infection; such biofilms can be bacterial, fungal, or mixed. In the past 15 years, there has been an increasing research effort into antimicrobial surfaces but the great majority of these publications present research on bacteria, with some reports also testing resistance to fungi. Very few studies have focused exclusively on antifungal surfaces. However, with increasing recognition of the importance of fungal infections to human health, particularly related to infections at biomaterials, it would seem that the interest in antifungal surfaces is disproportionately low. In studies of both bacteria and fungi, fungi tend to be the minor focus with hypothesized antibacterial mechanisms of action often generalized to also explain the antifungal effect. Yet bacteria and fungi represent two Distinct biological Domains and possess substantially different cellular physiology and structure. Thus it is questionable whether these generalizations are valid. Here we review the scientific literature focusing on surface coatings prepared with antifungal agents covalently attached to the biomaterial surface. We present a critical analysis of generalizations and their evidence. This review should be of interest to researchers of “antimicrobial” surfaces by addressing specific issues that are key to designing and understanding antifungal biomaterials surfaces and their putative mechanisms of action.     

In situ protein microarrays capable of real-time kinetics analysis based on surface plasmon resonance imaging

In recent years, in situ protein synthesis microarray technologies have enabled protein microarrays to be created on demand just before they are needed. In this paper, we utilized the TUS-TER immobilization technology to allow label-free detection with real-time kinetics of protein–protein interactions using surface plasmon resonance imaging (SPRi). We constructed an expression-ready plasmid DNA with a C-terminal TUS fusion tag to directionally immobilize the in situ synthesized recombinant proteins onto the surface of the biosensor. The expression plasmid was immobilized on the polyethylene imine-modified gold surface, which was then coupled with a cell-free expression system on the flow cell of the SPRi instrument. The expressed TUS fusion proteins bind on the surface via the immobilized TER DNA sequence with high affinity (∼3–7 × 10⁻¹³ M). The expression and immobilization of the recombinant in situ expressed proteins were confirmed by probing with specific antibodies. The present study shows a new low cost method for in situ protein expression microarrays that has the potential to study the kinetics of protein–protein interactions. These protein microarrays can be created on demand without the problems of stability associated with protein arrays used in the drug discovery and biomarker discovery fields.     

Biosensor analysis of anti-citrullinated protein/peptide antibody affinity

Anti-citrullinated protein/peptide antibodies (ACPAs) are detected in rheumatoid arthritis (RA) sera and because of their strict association with the disease are considered marker antibodies, probably endowed with pathogenic potential. Antibody affinity is one of the parameters affecting pathogenicity. Three diagnostic citrullinated peptides—viral citrullinated peptide 1 (VCP1) and VCP2 derived from Epstein–Barr virus (EBV)-encoded proteins and histone citrullinated peptide 1 (HCP1) derived from histone H4—were synthesized as tetrameric multiple antigen peptides and immobilized on sensor chips CM5 type in a Biacore T100 instrument. Specific binding of purified antibodies from RA patients to the three peptides was analyzed by surface plasmon resonance using two arginine-containing sequences as controls. Employing a 1:1 binding model for affinity constant calculation, ACPAs interacted with VCP1 and VCP2 with lower apparent affinity (10⁻⁶ M > K_D > 10⁻⁷ M) and interacted with HCP1 with higher apparent affinity (K_D = 10⁻⁸ M). The results indicate that the binding to citrullinated peptides is characterized by wide differences in affinity, with slower association and faster dissociation rates in the case of antibodies to viral citrullinated peptides as compared with antibodies specific for the histone peptide. This biosensor analysis shows the high cross-reactivity of purified ACPAs that bind other citrullinated peptides besides the one used for purification.