Identification of an exosite at the neutrophil elastase/alpha-1-antitrypsin interface.

Neutrophil elastase (NE) is released by activated neutrophils during an inflammatory response and exerts proteolytic activity on elastin and other extracellular matrix components. This protease is rapidly inhibited by the plasma serine protease inhibitor alpha-1-antitrypsin (AAT), and the importance of this protective activity on lung tissue is highlighted by the development of early onset emphysema in individuals with AAT deficiency. As a serpin, AAT presents a surface-exposed reactive centre loop (RCL) whose sequence mirrors the target protease specificity.

Reduction of Z alpha-1 antitrypsin polymers in human iPSC-hepatocytes and mice by LRRK2 inhibitors.

Background: Alpha-1 antitrypsin deficiency (A1ATD) is a life-threatening condition caused by the inheritance of the serpin family A member 1 "Z" genetic variant driving alpha-1 antitrypsin (AAT) protein misfolding in hepatocytes. There are no approved medicines for this disease.

Methods: We conducted a high-throughput image-based small molecule screen using patient-derived induced pluripotent stem cell-hepatocytes (iPSC-hepatocytes).

A novel pathological mutant reveals the role of torsional flexibility in the serpin breach in adoption of an aggregation-prone intermediate.

Mutants of alpha-1-antitrypsin cause the protein to self-associate and form ordered aggregates ('polymers') that are retained within hepatocytes, resulting in a predisposition to the development of liver disease. The associated reduction in secretion, and for some mutants, impairment of function, leads to a failure to protect lung tissue against proteases released during the inflammatory response and an increased risk of emphysema. We report here a novel deficiency mutation (Gly192Cys), that we name the Sydney variant, identified in a patient in heterozygosity with the Z allele (Glu342Lys).

Native and Ion Mobility Mass Spectrometry Characterization of Alpha 1 Antitrypsin Variants and Oligomers.

In this chapter, we describe a method for analyzing both recombinant and plasma-derived alpha 1 antitrypsin and its oligomers by means of native ion mobility mass spectrometry. Our experimental workflow can be applied to other variants of alpha 1 antitrypsin and its oligomers as well as being used to probe their interactions with small molecules in the gas phase.

Probing of the reactive center loop region of alpha-1-antitrypsin by mutagenesis predicts new type-2 dysfunctional variants.

Lung disease in alpha-1-antitrypsin deficiency (AATD) mainly results from insufficient control of the serine proteases neutrophil elastase (NE) and proteinase-3 due to reduced plasma levels of alpha-1-antitrypsin (AAT) variants. Mutations in the specificity-determining reactive center loop (RCL) of AAT would be predicted to minimally affect protein folding and secretion by hepatocytes but can impair anti-protease activity or alter the target protease. These properly secreted but dysfunctional 'type-2' variants would not be identified by common diagnostic protocols that are predicated on a reduction in circulating AAT.

Viral SERPINS-A Family of Highly Potent Immune-Modulating Therapeutic Proteins.

Serine protease inhibitors, SERPINS, are a highly conserved family of proteins that regulate serine proteases in the central coagulation and immune pathways, representing 2-10% of circulating proteins in the blood. Serine proteases form cascades of sequentially activated enzymes that direct thrombosis (clot formation) and thrombolysis (clot dissolution), complement activation in immune responses and also programmed cell death (apoptosis). Virus-derived serpins have co-evolved with mammalian proteases and serpins, developing into highly effective inhibitors of mammalian proteolytic pathways.

The under-appreciated world of the serpin family of serine proteinase inhibitors.

In the practice of medicine, many fundamental biological pathways that require tight on/off control, such as inflammation and circulatory homeostasis, are regulated by serine proteinases, but we rarely consider the unique protease inhibitors that, in turn, regulate these proteases. The serpins are a family of proteins with a shared tertiary structure, whose members largely act as serine protease inhibitors, found in all forms of life, ranging from viruses, bacteria, and archaea to plants and animals. These proteins represent up to 2-10% of proteins in the human blood and are the third most common protein family.

Comprehensive Clinical Diagnostic Pipelines Reveal New Variants in Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency (AATD) is an underdiagnosed disorder associated with mutations in the gene encoding alpha-1 antitrypsin (AAT). Severe AATD can manifest as pulmonary emphysema and progressive liver disease. Besides the most common pathogenic variants S (E264V) and Z (E342K), many rarer genetic variants of AAT have been found in patients and in the general population.

Dual reactivity disulfide bridging reagents; enabling new approaches to antibody fragment bioconjugation.

Disulfide bridging, also known as disulfide stapling, is a powerful strategy for the construction of site-selective protein bioconjugates. Here we describe the first examples of a new class of such reagents, containing a 'stable-labile' design. These dual-reactive reagents are designed to form a stable bond to one cysteine and a labile bond to the second; resulting in a robust attachment to the protein with one end of the bridge, whilst the other end serves as a reactive handle for subsequent bioconjugation.

Keratin 8 is a scaffolding and regulatory protein of ERAD complexes.

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway.

The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant.

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap.

Scaling Concepts in Serpin Polymer Physics.

α1-Antitrypsin is a protease inhibitor belonging to the serpin family. Serpin polymerisation is at the core of a class of genetic conformational diseases called serpinopathies. These polymers are known to be unbranched, flexible, and heterogeneous in size with a beads-on-a-string appearance viewed by negative stain electron microscopy.

The development of highly potent and selective small molecule correctors of Z α-antitrypsin misfolding.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.

Repeated binge ethanol drinking enhances electrical activity of central amygdala corticotropin releasing factor neurons in vivo.

Binge ethanol drinking is an increasingly problematic component of alcohol use disorder costing the United States approximately over $150 billion every year and causes progressive neuroplasticity alterations in numerous brain regions. However, the precise nature or machinery that underlies binge drinking has not yet been elucidated. Corticotropin releasing factor (CRF) neurons in the central amygdala (CeA) are thought to modulate binge drinking, but the specific circuit mechanisms remain poorly understood.

Conversion of the death inhibitor ARC to a killer activates pancreatic β cell death in diabetes.

Loss of insulin-secreting pancreatic β cells through apoptosis contributes to the progression of type 2 diabetes, but underlying mechanisms remain elusive. Here, we identify a pathway in which the cell death inhibitor ARC paradoxically becomes a killer during diabetes. While cytoplasmic ARC maintains β cell viability and pancreatic architecture, a pool of ARC relocates to the nucleus to induce β cell apoptosis in humans with diabetes and several pathophysiologically distinct mouse models.

Development of a small molecule that corrects misfolding and increases secretion of Z α -antitrypsin.

Severe α -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α -antitrypsin. The lead compound blocks Z α -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α -antitrypsin threefold in an iPSC model of disease.

High-resolution ex vivo NMR spectroscopy of human Z α-antitrypsin.

Genetic mutations predispose the serine protease inhibitor α-antitrypsin to misfolding and polymerisation within hepatocytes, causing liver disease and chronic obstructive pulmonary disease. This misfolding occurs via a transiently populated intermediate state, but our structural understanding of this process is limited by the instability of recombinant α-antitrypsin variants in solution. Here we apply NMR spectroscopy to patient-derived samples of α-antitrypsin at natural isotopic abundance to investigate the consequences of disease-causing mutations, and observe widespread chemical shift perturbations for methyl groups in Z AAT (E342K).

The structural basis for Z α-antitrypsin polymerization in the liver.

The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α-antitrypsin as "polymer" chains in liver tissue. No detailed structural analysis has been performed of this material.

The molecular species responsible for α -antitrypsin deficiency are suppressed by a small molecule chaperone.

The formation of ordered Z (Glu342Lys) α -antitrypsin polymers in hepatocytes is central to liver disease in α -antitrypsin deficiency. In vitro experiments have identified an intermediate conformational state (M*) that precedes polymer formation, but this has yet to be identified in vivo. Moreover, the mechanism of polymer formation and their fate in cells have been incompletely characterised.

Intrahepatic heteropolymerization of M and Z alpha-1-antitrypsin.

The α-1-antitrypsin (or alpha-1-antitrypsin, A1AT) Z variant is the primary cause of severe A1AT deficiency and forms polymeric chains that aggregate in the endoplasmic reticulum of hepatocytes. Around 2%-5% of Europeans are heterozygous for the Z and WT M allele, and there is evidence of increased risk of liver disease when compared with MM A1AT individuals. We have shown that Z and M A1AT can copolymerize in cell models, but there has been no direct observation of heteropolymer formation in vivo.

Lanthanides compete with calcium for binding to cadherins and inhibit cadherin-mediated cell adhesion.

Lanthanides are rare-earth metals with a broad range of applications in biological research and medicine. In addition to their unique magnetic and spectroscopic properties, lanthanides are also effective mimics of calcium and can stimulate or inhibit the function of calcium-binding proteins. Cadherins are a large family of calcium-binding proteins that facilitate cell adhesion and play key roles in embryo development, tissue homeostasis and tumour metastasis.

Characterisation of a type II functionally-deficient variant of alpha-1-antitrypsin discovered in the general population.

Lung disease in alpha-1-antitrypsin deficiency (AATD) results from dysregulated proteolytic activity, mainly by neutrophil elastase (HNE), in the lung parenchyma. This is the result of a substantial reduction of circulating alpha-1-antitrypsin (AAT) and the presence in the plasma of inactive polymers of AAT. Moreover, some AAT mutants have reduced intrinsic activity toward HNE, as demonstrated for the common Z mutant, as well as for other rarer variants.

In Vitro Approaches for the Assessment of Serpin Polymerization.

Serpin polymerization is the result of end-to-end ordered aggregation of serpin monomers into linear unbranched chains. This change in molecular state represents the basis of several conformational diseases with pathological gain-of-function and loss-of-function phenotypes, termed serpinopathies. Tools that enable quantification and characterization of polymer formation are therefore important to the study of serpin behavior in this pathophysiological context.

Probing the folding pathway of a consensus serpin using single tryptophan mutants.

Conserpin is an engineered protein that represents the consensus of a sequence alignment of eukaryotic serpins: protease inhibitors typified by a metastable native state and a structurally well-conserved scaffold. Previously, this protein has been found to adopt a native inhibitory conformation, possess an atypical reversible folding pathway and exhibit pronounced resistance to inactivation. Here we have designed a version of conserpin, cAT, with the inhibitory specificity of α-antitrypsin, and generated single-tryptophan variants to probe its folding pathway in more detail.

Electrophoresis- and FRET-Based Measures of Serpin Polymerization.

Many serpinopathies, including alpha-1 antitrypsin (A1AT) deficiency, are associated with the formation of unbranched polymer chains of mutant serpins. In vivo, this deficiency is the result of mutations that cause kinetic or thermodynamic destabilization of the molecule. However, polymerization can also be induced in vitro from mutant or wild-type serpins under destabilizing conditions.