Development of Lyophilized Eukaryotic Cell-Free Protein Expression System Based on .

Eukaryotic cell-free protein expression systems enable rapid production of recombinant multidomain proteins in their functional form. A cell-free system based on the rapidly growing protozoan (LTE) has been extensively used for protein engineering and analysis of protein interaction networks. However, like other eukaryotic cell-free systems, LTE deteriorates at ambient temperatures and requires deep freezing for transport and storage.

Reconstitution and Analysis of SARS-CoV-2/Host Protein-Protein Interactions.

The emergence of viral threats such as Ebola, ZIKA, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) requires a rapid and efficient approach for elucidating mechanisms of pathogenesis and development of therapeutics. In this context, cell-free protein synthesis (CFPS) holds a promise to resolve the bottlenecks of multiplexed protein production and interaction analysis among host and pathogen proteins. Here, we applied a eukaryotic CFPS system based on extract (LTE) protein expression in combination with AlphaLISA proximity-based protein interaction technology to identify intraviral and viral-human protein interactions of SARS-CoV-2 virus that can potentially be targeted by the existing or novel antiviral therapeutics.

HDAC7 is an immunometabolic switch triaging danger signals for engagement of antimicrobial versus inflammatory responses in macrophages.

The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1β production.

Engineering and exploiting synthetic allostery of NanoLuc luciferase.

Allostery enables proteins to interconvert different biochemical signals and form complex metabolic and signaling networks. We hypothesize that circular permutation of proteins increases the probability of functional coupling of new N- and C- termini with the protein's active center through increased local structural disorder. To test this we construct a synthetically allosteric version of circular permutated NanoLuc luciferase that can be activated through ligand-induced intramolecular non-covalent cyclisation.

Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system.

Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping. While in vitro translation systems are well suited for fast and multiplexed polypeptide prototyping, they suffer from misfolding, aggregation and disulfide-bond scrambling of the translated products. Here we propose that efficient folding of in vitro produced disulfide-rich peptides and proteins can be achieved if performed in an aggregation-free and thermodynamically controlled folding environment.

Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle.

The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems, mouse and zebrafish. In both models, Cavin4 localized to T-tubules, and loss of Cavin4 resulted in aberrant T-tubule maturation.

An ancient viral epidemic involving host coronavirus interacting genes more than 20,000 years ago in East Asia.

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has emphasized the vulnerability of human populations to novel viral pressures, despite the vast array of epidemiological and biomedical tools now available. Notably, modern human genomes contain evolutionary information tracing back tens of thousands of years, which may help identify the viruses that have impacted our ancestors-pointing to which viruses have future pandemic potential. Here, we apply evolutionary analyses to human genomic datasets to recover selection events involving tens of human genes that interact with coronaviruses, including SARS-CoV-2, that likely started more than 20,000 years ago.

Class IIa Histone Deacetylases Drive Toll-like Receptor-Inducible Glycolysis and Macrophage Inflammatory Responses via Pyruvate Kinase M2.

Histone deacetylases (HDACs) drive innate immune cell-mediated inflammation. Here we identify class IIa HDACs as key molecular links between Toll-like receptor (TLR)-inducible aerobic glycolysis and macrophage inflammatory responses. A proteomic screen identified the glycolytic enzyme pyruvate kinase M isoform 2 (Pkm2) as a partner of proinflammatory Hdac7 in murine macrophages.

Identification of intracellular cavin target proteins reveals cavin-PP1alpha interactions regulate apoptosis.

Caveolae are specialized domains of the plasma membrane. Formation of these invaginations is dependent on the expression of Caveolin-1 or -3 and proteins of the cavin family. In response to stress, caveolae disassemble and cavins are released from caveolae, allowing cavins to potentially interact with intracellular targets.

Adaption of the Leishmania Cell-Free Expression System to High-Throughput Analysis of Protein Interactions.

In this chapter, we present methods for adapting the eukaryotic cell-free expression system based on Leishmania tarentolae to high-throughput analysis of protein interactions. Specifically, we present a lysate optimization technique that minimizes the amount of unwanted premature termination products while balancing protein expression level and protein aggregation. Finally, we present methods for adapting the Leishmania cell-free system to the AlphaLISA-based protein interaction assay.

Glycosylation, an effective synthetic strategy to improve the bioavailability of therapeutic peptides.

Glycosylation of peptides is a promising strategy for modulating the physicochemical properties of peptide drugs and for improving their absorption through biological membranes. This review highlights various methods for the synthesis of glycoconjugates and recent progress in the development of glycosylated peptide therapeutics. Furthermore, the impacts of glycosylation in overcoming the existing barriers that restrict oral and brain delivery of peptides are described herein.

Evaluation of the Biological Properties and the Enzymatic Stability of Glycosylated Luteinizing Hormone-Releasing Hormone Analogs.

The enzymatic stability, antitumor activity, and gonadotropin stimulatory effects of glycosylated luteinizing hormone-releasing hormone (LHRH) analogs were investigated in this study. Conjugation of carbohydrate units, including lactose (Lac), glucose (GS), and galactose (Gal) to LHRH peptide protected the peptide from proteolytic degradation and increased the peptides' half-lives in human plasma, rat kidney membrane enzymes, and liver homogenate markedly. Among all seven modified analogs, compound 1 (Lac-[Q(1)][w(6)]LHRH) and compound 6 (GS(4)-[w(6)]LHRH) were stable in human plasma during 4 h of experiment.

The transport and efflux of glycosylated luteinising hormone-releasing hormone analogues in caco-2 cell model: contributions of glucose transporters and efflux systems.

Luteinising hormone-releasing hormone (LHRH) analogues have wide therapeutic applications in the treatment of prostate cancers and endocrine disorders. The structure of LHRH was modified using a glycosylation strategy to increase the permeability of the peptide across biological membranes. Lactose, galactose and glucose units were coupled to LHRH peptide, and the impact of glucose transporters, GLUT2 and SGLT1, was investigated in the transport of the analogues.

Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH).

Luteinizing hormone-releasing hormone (LHRH) analogues are used extensively for the treatment of various hormone-dependent diseases. However, none of the currently marketed derivatives can be administered orally. Modification of peptide sequences by attachment of carbohydrate moieties is a promising strategy that may increase the metabolic stability of the target peptide and enhance its transport across cell membranes, subsequently improving peptide bioavailability.