DNA sequencing with chain terminating inhibitors

Autoimmune diseases are immune disorders characterized by T cell hyperactivity and B cell overstimulation leading to overproduction of autoantibodies. Although the pathogenesis of various autoimmune diseases remains to be elucidated, environmental factors have been thought to contribute to the initiation and maintenance of auto-respond inflammation. Toll-like receptors (TLRs) are pattern recognition receptors belonging to innate immunity that recognize and defend invading microorganisms. Besides these exogenous pathogen-associated molecular patterns, TLRs can also bind with damage-associated molecular patterns produced under strike or by tissue damage or cells apoptosis. It is believed that TLRs build a bridge between innate immunity and autoimmunity. There are five adaptors to TLRs including MyD88, TRIF, TIRAP/MAL, TRAM, and SARM. Upon activation, TLRs recruit specific adaptors to initiate the downstream signaling pathways leading to the production of inflammatory cytokines and chemokines. Under certain circumstances, ligation of TLRs drives to aberrant activation and unrestricted inflammatory responses, thereby contributing to the perpetuation of inflammation in autoimmune diseases. In the past, most studies focused on the intracellular TLRs, such as TLR3, TLR7, and TLR9, but recent studies reveal that cell surface TLRs, especially TLR2 and TLR4, also play an essential role in the development of autoimmune diseases and afford multiple therapeutic targets. In this review, we summarized the biological characteristics, signaling mechanisms of TLR2/4, the negative regulators of TLR2/4 pathway, and the pivotal function of TLR2/4 in the pathogenesis of autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, psoriasis, multiple sclerosis, and autoimmune diabetes.

TLR2 and TLR4 in Autoimmune Diseases: a Comprehensive Review

SAA is a major acute-phase protein produced in large quantity during APR. The rise of SAA concentration in blood circulation during APR has been a clinical marker for active inflammation. In the past decade, research has been conducted to determine whether SAA plays an active role during inflammation and if so, how it influences the course of inflammation. These efforts have led to the discovery of cytokine-like activities of rhSAA, which is commercially available and widely used in most of the published studies. SAA activates multiple receptors, including the FPR2, the TLRs TLR2 and TLR4, the scavenger receptor SR-BI, and the ATP receptor P2X7. More recent studies have shown that SAA not only activates transcription factors, such as NF-kB, but also plays a role in epigenetic regulation through a MyD88IRF4-Jmjd3 pathway. It is postulated that the activation of these pathways leads to induced expression of proinflammatory factors and a subset of proteins expressed by the M2 macrophages. These functional properties set SAA apart from well-characterized inflammatory factors, such as LPS and TNF-a, suggesting that it may play a homeostatic role during the course of inflammation. Ongoing and future studies are directed to addressing unresolved issues, including the difference between rSAA and native SAA isoforms and the exact functions of SAA in physiologic and pathologic settings. J. Leukoc. Biol. 98: 923–929; 2015.

https://jlb.onlinelibrary.wiley.com/doi/pdf/10.1189/jlb.3VMR0315-080R

Emerging functions of serum amyloid A in inflammation

Scleroderma is a progressive autoimmune disease affecting multiple organs. Fibrosis, the hallmark of scleroderma, represents transformation of self-limited wound healing into a deregulated self-sustaining process. The factors responsible for maintaining persistent fibroblast activation in scleroderma and other conditions with chronic fibrosis are not well understood. Toll-like receptor 4 (TLR4) and its damage-associated endogenous ligands are implicated in immune and fibrotic responses. We now show that fibronectin extra domain A (FnEDA) is an endogenous TLR4 ligand markedly elevated in the circulation and lesional skin biopsies from patients with scleroderma, as well as in mice with experimentally induced cutaneous fibrosis. Synthesis of FnEDA was preferentially stimulated by transforming growth factor–β in normal fibroblasts and was constitutively up-regulated in scleroderma fibroblasts. Exogenous FnEDA was a potent stimulus for collagen production, myofibroblast differentiation, and wound healing in vitro and increased the mechanical stiffness of human organotypic skin equivalents. Each of these profibrotic FnEDA responses was abrogated by genetic, RNA interference, or pharmacological disruption of TLR4 signaling. Moreover, either genetic loss of FnEDA or TLR4 blockade using a small molecule mitigated experimentally induced cutaneous fibrosis in mice. These observations implicate the FnEDA-TLR4 axis in cutaneous fibrosis and suggest a paradigm in which aberrant FnEDA accumulation in the fibrotic milieu drives sustained fibroblast activation via TLR4. This model explains how a damage-associated endogenous TLR4 ligand might contribute to converting self-limited tissue repair responses into intractable fibrogenesis in chronic conditions such as scleroderma. Disrupting sustained TLR4 signaling therefore represents a potential strategy for the treatment of fibrosis in scleroderma.

/pdf/fibronectineda-promotes-chronic-cutaneous-fibrosis-through-3p9i9l5s2a.pdf

FibronectinEDA Promotes Chronic Cutaneous Fibrosis Through Toll-like Receptor Signaling

Production of fine heterologus pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved and enzymes lack detailed characterization An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes We demonstrate this concept using P450BM3 from Bacillus megaterium Using a computer model, we illustrate how key P450BM3 activ site mutations enable binding of non-native substrate amorphadiene, incorporating these mutations into P450BM3 enabled the selective oxidation of amorphadiene arteminsinic-11s,12-epoxide, at titers of 250 mg L 1 in E coli We also demonstrate high-yeilding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high value anti-malarial drug artemisinin

A Novel Semi-biosynthetic Route for Artemisinin Production Using Engineered Substrate-Promiscuous P450BM3

Enzymes are attractive tools for synthetic applications. To be viable for industrial use, enzymes need sufficient stability towards the desired reaction conditions such as high substrate and cosolvent concentration, non-neutral pH and elevated temperatures. Thermal stability is an attractive feature not only because it allows for protein purification by thermal treatment and higher process temperatures but also due to the associated higher stability against other destabilising factors. Therefore, high-throughput screening (HTS) methods are desirable for the identification of thermostable biocatalysts by discovery from nature or by protein engineering but current methods have low throughput and require time-demanding purification of protein samples. We found that nanoscale differential scanning fluorimetry (nanoDSF) is a valuable tool to rapidly and reliably determine melting points of native proteins. To avoid intrinsic problems posed by crude protein extracts, hypotonic extraction of overexpressed protein from bacterial host cells resulted in higher sample quality and accurate manual determination of several hundred melting temperatures per day. We have probed the use of nanoDSF for HTS of a phylogenetically diverse aldolase library to identify novel thermostable enzymes from metagenomic sources and for the rapid measurements of variants from saturation mutagenesis. The feasibility of nanoDSF for the screening of synthetic reaction conditions was proved by studies of cosolvent tolerance, which showed protein melting temperature to decrease linearly with increasing cosolvent concentration for all combinations of six enzymes and eight water-miscible cosolvents investigated, and of substrate affinity, which showed stabilisation of hexokinase by sugars in the absence of ATP cofactor. ENZYMES: Alcohol dehydrogenase (NADP+ ) (EC 1.1.1.2), transketolase (EC 2.2.1.1), hexokinase (EC 2.7.1.1), 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), fructose-6-phosphate aldolase (EC 4.1.2.n).

nanoDSF as screening tool for enzyme libraries and biotechnology development.

Finding faster and simpler ways to screen protein sequence space to enable the identification of new biocatalysts for asymmetric synthesis remains both a challenge and a rate-limiting step in enzyme discovery. Biocatalytic strategies for the synthesis of chiral amines are increasingly attractive and include enzymatic asymmetric reductive amination, which offers an efficient route to many of these high-value compounds. Here we report the discovery of over 300 new imine reductases and the production of a large (384 enzymes) and sequence-diverse panel of imine reductases available for screening. We also report the development of a facile high-throughput screen to interrogate their activity. Through this approach we identified imine reductase biocatalysts capable of accepting structurally demanding ketones and amines, which include the preparative synthesis of N-substituted β-amino ester derivatives via a dynamic kinetic resolution process, with excellent yields and stereochemical purities.

Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination.

Systemic sclerosis is an autoimmune idiopathic connective tissue disease, characterized by vasculopathy, inflammation and fibrosis. There appears to be a link between inflammation and fibrosis, although the exact nature of the relationship is unknown. Serum amyloid A (SAA) is an acute-phase protein that is elevated up to 1000-fold in times of infection or inflammation. This acute-phase reactant, as well as being a marker of inflammation, may initiate signals in a cytokine-like manner, possibly through toll-like receptors (TLRs) promoting inflammation. This study addressed the role of SAA in initiating interleukin-6 (IL-6) production in dermal fibroblasts and the role of TLR2 in this system. We show that SAA induces IL-6 secretion in healthy dermal fibroblasts and that blockade of TLR2 with a neutralizing antibody to TLR2 or specific small interfering RNA attenuated the SAA-induced IL-6 secretion and that this was also mediated through the TLR adaptor protein IL-1 receptor-associated kinase 4. The effect is nuclear factor-κB-mediated because blockade of nuclear factor-κB reduced the induction. We also demonstrate that dermal fibroblasts express TLR2; this is functional and over-expressed in the fibroblasts of patients with systemic sclerosis. Taken together these data suggest that SAA is a danger signal that initiates IL-6 signalling in systemic sclerosis via enhanced TLR2 signalling.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/imm.12260

Serum amyloid A induces interleukin‐6 in dermal fibroblasts via Toll‐like receptor 2, interleukin‐1 receptor‐associated kinase 4 and nuclear factor‐κB

Enantioenriched 2-aryl azepanes and 2-arylbenzazepines were generated biocatalytically by asymmetric reductive amination using imine reductases or by deracemization using monoamine oxidases. The amines were converted to the corresponding N′-aryl ureas, which rearranged on treatment with base with stereospecific transfer of the aryl substituent to the 2-position of the heterocycle via a configurationally stable benzyllithium intermediate. The products are previously inaccessible enantioenriched 2,2-disubstituted azepanes and benzazepines.

/pdf/chemoenzymatic-synthesis-of-substituted-azepanes-by-26z4low4rf.pdf

Chemoenzymatic Synthesis of Substituted Azepanes by Sequential Biocatalytic Reduction and Organolithium-Mediated Rearrangement.

Accumulative evidence demonstrates the crucial role of evolutionary conserved Toll-like receptors (TLRs) in identifying microbial or viral compounds. TLRs are also able to recognise endogenous molecules which are released upon cell damage or stress and have been shown to play a key role in numerous autoimmune diseases including systemic sclerosis (SSc). A classic feature of SSc, is vascular injury manifested as Raynaud's phenomenon and ischaemia of the skin, resulting in the release of endogenous TLR ligands during inflammation and local tissue damage. These locally released TLR ligands bind TLRs possibly complexed to autoantibodies, and initiate intracellular signalling pathways and may be one of the mechanisms that initiate and drive autoimmunity and subsequent fibrosis. Activation of the immune system results in interferon (IFN) sensitive gene transcription. There is also an IFN gene signature in SSc peripheral blood. TLRs may represent the link between immune activation, common in SSc, and tissue fibrosis. Therefore, a better understanding of the mechanisms of TLR-mediated pathogenesis and therapies targeting individual TLRs, may provide a more specific approach of treating multi-systemic autoimmune diseases. This review aims to integrate the current knowledge of TLR function in the autoimmune disorders with particular emphasis on SSc. We suggest the TLR system as a new therapeutic target.

James Finnigan

Papers

nanoDSF as screening tool for enzyme libraries and biotechnology development.

Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination.

Serum amyloid A induces interleukin‐6 in dermal fibroblasts via Toll‐like receptor 2, interleukin‐1 receptor‐associated kinase 4 and nuclear factor‐κB

Chemoenzymatic Synthesis of Substituted Azepanes by Sequential Biocatalytic Reduction and Organolithium-Mediated Rearrangement.

Role of toll-like receptors in systemic sclerosis