Hydrogen-Deuterium Exchange Epitope Mapping of Glycosylated Epitopes Enabled by Online Immobilized Glycosidase.

Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is widely used for monoclonal antibody (mAb) epitope mapping, which aids in the development of therapeutic mAbs and vaccines, as well as enables the understanding of viral immune evasion. Numerous mAbs are known to recognize N-glycosylated epitopes and to bind in close proximity to an -glycan site; however, glycosylated protein sites are typically obscured from HDX detection as a result of the inherent heterogeneity of glycans. To overcome this limitation, we covalently immobilized the glycosidase PNGase Dj on a solid resin and incorporated it into an online HDX-MS workflow for post-HDX deglycosylation.

An anti-ANGPTL3/8 antibody decreases circulating triglycerides by binding to a LPL-inhibitory leucine zipper-like motif.

Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition.

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19).

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

Unlabelled: SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity.

Computational stabilization of T cell receptors allows pairing with antibodies to form bispecifics.

Recombinant T cell receptors (TCRs) can be used to redirect naïve T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Here, we use molecular modeling to identify mutations in the TCR constant domains (Cα/Cβ) that increase the unfolding temperature of Cα/Cβ by 20 °C, improve the expression of four separate α/β TCRs by 3- to 10-fold, and improve the assembly and stability of TCRs with poor intrinsic stability. The stabilizing mutations rescue the expression of TCRs destabilized through variable domain mutation.

Modulation of the Biophysical Properties of Bifunctional Antibodies as a Strategy for Mitigating Poor Pharmacokinetics.

Interest in the development of bi- or multispecific antibody (BsAbs)-based biotherapeutics is growing rapidly due to their inherent ability to interact with many targets simultaneously, thereby potentially protracting their functionality relative to monoclonal antibodies (mAbs). Biophysical property assays have been used to improve the probability of clinical success for various mAb therapeutics; however, there is a paucity of such data for BsAbs. This work evaluates a fusion of an IgG with an isolated protein domain (deemed ECD) and serves to understand how molecular architecture influences biophysical and biochemical properties and, in turn, how these relate to drug disposition.

The folding unit of phosphofructokinase-2 as defined by the biophysical properties of a monomeric mutant.

Escherichia coli phosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N2 ↔ 2I ↔ 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a flattened β-barrel. Although isolated monomers of E.

How the ankyrin and SOCS box protein, ASB9, binds to creatine kinase.

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. The ASB proteins function as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that specifically transfer ubiquitin to cellular proteins targeting them for degradation by the proteasome. ASB9 binds to creatine kinase (CK) and targets it for degradation; however, the way in which ASB9 interacts with CK is not yet known.

The phylogenetics of dynamics in DNA clamp proteins.

Using HXMS to measure protein dynamics, Fang and colleagues (in this issue of Structure) probe the dynamics of processivity clamp proteins from all kingdoms of life. The proteins have similar structures but divergent sequences. Their results show conserved dynamics correlating with primary functions and divergent dynamics for divergent functions.

Predicted disorder-to-order transition mutations in IκBα disrupt function.

IκBα inhibits the transcription factor, NFκB, by forming a very tightly bound complex in which the ankyrin repeat domain (ARD) of IκBα interacts primarily with the dimerization domain of NFκB. The first four ankyrin repeats (ARs) of the IκBα ARD are well-folded, but the AR5-6 region is intrinsically disordered according to amide H/D exchange and protein folding/unfolding experiments. We previously showed that mutations towards the consensus sequence for stable ankyrin repeats resulted in a "prefolded" mutant.

Effects of linalool on inflammation, matrix accumulation and podocyte loss in kidney of streptozotocin-induced diabetic rats.

Context: This study aimed to evaluate the renoprotective action of linalool (LIN) on streptozotocin (STZ)-induced diabetic rats.

Objective: The pathological changes in diabetic nephropathy (DN) include oxidative stress, renal injury, matrix accumulation and podocyte abnormalities. We investigated the renoprotective actions of LIN, a monoterpene alcohol, present in herbal essential oils in STZ-induced diabetic rats with renal injury.

Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins.

Amide hydrogen/deuterium exchange detected by mass spectrometry (HXMS) is seeing wider use for the identification of intrinsically disordered parts of proteins. In this review, we discuss examples of how discovery of intrinsically disordered regions and their removal can aid in structure determination, biopharmaceutical quality control, the characterization of how post-translational modifications affect weak structuring of disordered regions, the study of coupled folding and binding, and the characterization of amyloid formation. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

Specificity of HCPTP variants toward EphA2 tyrosines by quantitative selected reaction monitoring.

EphA2 receptor tyrosine kinase and the human cytoplasmic protein tyrosine phosphatase (HCPTP) are overexpressed in a number of epithelial cancers. Overexpressed EphA2 in these cancers shows a significant decrease in phosphotyrosine content which results in suppression of receptor signaling and endocytosis and an increase in metastatic potential. The decreased phosphotyrosine content of EphA2 has been associated with decreased contact with its ligand, ephrin A1 and dephosphorylation by HCPTP.

Use of selected reaction monitoring data for label-free quantification of protein modification stoichiometry.

Quantification of protein and PTM abundance in biological samples is an important component of proteomic studies. Label-free methods for quantification using MS are attractive because they are simple to implement and applicable to any experimental system. We demonstrate that PTM stoichiometry can be accurately measured using label-free quantification and selected reaction monitoring.

Identification of novel inhibitors for a low molecular weight protein tyrosine phosphatase via virtual screening.

The human cytoplasmic protein tyrosine phosphatase (HCPTP) has been identified as a potential target for inhibition in order to downregulate metastatic transformation in several human epithelial cancers such as breast, prostate and colon cancer. Docking with two scoring functions on both isoforms of HCPTP was employed as an initial virtual screen to identify potential inhibitors. Compounds identified as potential inhibitors via this in silico screen were subjected to kinetic analysis in order to validate their selection as improved inhibitors.

Tobacco protein separation by aqueous two-phase extraction.

Tobacco has long been considered as a host to produce large quantity of high-valued recombinant proteins. However, dealing with large quantities of biomass is a challenge for downstream processing. Aqueous two-phase extraction (ATPE) has been widely used in purifying proteins from various sources.