Modifying the Substrate Specificity of Carcinoscorpius rotundicauda Serine Protease Inhibitor Domain 1 to Target Thrombin

Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC50 of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases

Capturing the Fantastic Voyage of Monocytes Through Time and Space

Monocytes are a subset of cells that are categorized together with dendritic cells (DCs) and macrophages in the mononuclear phagocyte system (MPS). Despite sharing several phenotypic and functional characteristics with MPS cells, monocytes are unique cells with the ability to function as both precursor and effector cells in their own right. Before the development of hematopoietic stem cells (HSCs) in utero, monocytes are derived from erythro-myeloid precursors (EMPs) in the fetal liver that are important for populating the majority of tissue resident macrophages. After birth, monocytes arise from bone marrow (BM)-derived HSCs and are released into the circulation upon their maturation, where they survey peripheral tissues and maintain endothelial integrity. Upon sensing of microbial breaches or inflammatory stimuli, monocytes migrate into tissues where their plasticity allows them to differentiate into cells that resemble macrophages or DCs according to the environmental niche. Alternatively, they may also migrate into tissues in the absence of inflammation and remain in an undifferentiated state where they perform homeostatic roles. As monocytes are typically on the move, the availability of intravital imaging approaches has provided further insights into their trafficking patterns in distinct tissue compartments. In this review, we outline the importance of understanding their functional behavior in the context of tissue compartments, and how these studies may contribute towards improved vaccine and future therapeutic strategies

Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin

Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation

Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy

Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence

A Computational and Experimental Study of the Regulatory Mechanisms of the Complement System

The complement system is key to innate immunity and its activation is necessary for the clearance of bacteria and apoptotic cells. However, insufficient or excessive complement activation will lead to immune-related diseases. It is so far unknown how the complement activity is up- or down- regulated and what the associated pathophysiological mechanisms are. To quantitatively understand the modulatory mechanisms of the complement system, we built a computational model involving the enhancement and suppression mechanisms that regulate complement activity. Our model consists of a large system of Ordinary Differential Equations (ODEs) accompanied by a dynamic Bayesian network as a probabilistic approximation of the ODE dynamics. Applying Bayesian inference techniques, this approximation was used to perform parameter estimation and sensitivity analysis. Our combined computational and experimental study showed that the antimicrobial response is sensitive to changes in pH and calcium levels, which determines the strength of the crosstalk between CRP and L-ficolin. Our study also revealed differential regulatory effects of C4BP. While C4BP delays but does not decrease the classical complement activation, it attenuates but does not significantly delay the lectin pathway activation. We also found that the major inhibitory role of C4BP is to facilitate the decay of C3 convertase. In summary, the present work elucidates the regulatory mechanisms of the complement system and demonstrates how the bio-pathway machinery maintains the balance between activation and inhibition. The insights we have gained could contribute to the development of therapies targeting the complement system.Singapore. Ministry of Education (Grant T208B3109)Singapore. Agency for Science, Technology and Research (BMRC 08/1/21/19/574)Singapore-MIT Alliance (Computational and Systems Biology Flagship Project)Swedish Research Counci

International Conference on Pharmacy and Pharma Networks on “Recent Innovations and Advs in Pharmacy and Pharma Networks”

International Conference on Pharmacy and Pharma Network

The Genetic Engineering and Development of rFC for Endotoxin Detection

Endotoxin Conference - Keynote Lectur

rFC - from genetic engineering to biomedical /Industrial applications in endotoxin detection

2018 Parenteral Drug Association Endotoxins WorkshopUnited State

rFC: Engineered for Endotoxin Test

PharmaLab 202