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α1-Antitrypsin, the archetypal member of the serpin superfamily, is a metastable protein prone to polymerization when exposed to stressors such as elevated temperature, low denaturant concentrations or through the presence of deleterious mutations which, in a physiological context, are often associated with disease. Experimental evidence suggests that a1 Antitrypsin can polymerize via several alternative mechanisms in vitro. In these polymerization mechanisms different parts of the molecule are proposed to undergo conformational change. Both strand 5 and helix I are proposed to adopt different conformations when forming the various polymers, and possess a number of highly conserved residues however their role in the folding and misfolding of α1-Antitrypsin has never been examined. We have therefore created a range of α1 Antitypsin variants in order to explore the role of these conserved residues in serpin folding, misfolding, stability and function. Our data suggest that key residues in helix I mediate efficient folding from the folding intermediate and residues in strand 5A ensure native state stability in order to prevent misfolding. Additionally, our data indicate that helix I is involved in the inhibitory process and that both structural elements undergo differing conformational rearrangements during unfolding and misfolding. These findings suggest that the ability of α1-Antitrypsin to adopt different types of polymers under different denaturing conditions may be due to subtle conformational differences in the transiently populated structures adopted prior to the I and M* states. [ABSTRACT FROM AUTHOR]

Abstract: The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α1-antitrypsin (α1AT) deficiency cases. It leads to increased polymerization of the serpin α1AT during its synthesis and in circulation. It has been proposed that the Z mutation results in a conformational change within the folded state of antitrypsin that enhances its polymerization. In order to localize the conformational change, we have created two single tryptophan mutants of Z α1AT and analyzed their fluorescence properties. α1AT contains two tryptophan residues that are located in distinct regions of the molecule: Trp194 at the top of β-sheet A and Trp238 on β-sheet B. We have replaced each tryptophan residue individually with a phenylalanine in order to study the local environment of the remaining tryptophan residue in both M and Z α1AT. A detailed fluorescence spectroscopic analysis of each mutant was carried out, and we detected differences in the emission spectrum, the Stern–Volmer constant for potassium iodide quenching and the anisotropy of only Trp194 in Z α1AT compared to M α1AT. Our data reveal that the Z mutation results in a conformational change at the top of β-sheet A but does not affect the structural integrity of β-sheet B. [Copyright &y& Elsevier]

Abstract: The serpinopathies encompass a large number of diseases caused by inappropriate conformational change and self-association (polymerization) of a serpin (serine proteinase inhibitor) molecule. The most common serpinopathy is α1-antitrypsin (α1AT) deficiency, which is associated with an increased risk for liver cirrhosis, hepatocellular carcinoma and early-onset emphysema. The Z variant of α1AT, which accounts for 95% of all cases of α1AT deficiency, polymerizes during synthesis and after secretion. Here, we show using intrinsic and extrinsic fluorescence probes that Z α1AT exists in a non-native conformation. We examined the thermodynamic stability by transverse urea gradient gel electrophoresis, thermal denaturation and equilibrium guanidine hydrochloride unfolding and found that, despite structural differences between the two proteins, wild-type α1AT and Z α1AT display similar unfolding pathways and thermodynamic stabilities. Far-UV circular dichroism and bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt) fluorescence suggest that the intermediate ensembles formed during unfolding of wild-type α1AT and Z α1AT are characterized by similar structural features. Kinetic analysis of the unfolding transition showed that Z α1AT unfolds at least 1.5-fold faster than the wild type. The biological implications of these data are discussed. [Copyright &y& Elsevier]

Protein aggregation is the cause of several human diseases. Understanding the molecular mechanisms involved in protein aggregation requires knowledge of the kinetics and structures populated during the reaction. Arguably, the best structurally characterized misfolding reaction is that of α1-antitrypsin. α1-Antitrypsin misfolding leads to both liver disease and emphysema and affect approximately 1 in 2000 of the population. This review will focus on the mechanism of α1-antitrypsin misfolding and the development of potential therapeutic strategies. © 2008 IUBMB IUBMB Life, 61(1): 1–5, 2009 [ABSTRACT FROM AUTHOR]

Abstract: α1-Antitrypsin (α1AT), the most abundant proteinase inhibitor circulating in the blood, protects extracellular matrix proteins of the lung against proteolytic destruction by neutrophil elastase. α1AT deficiency predisposes patients to emphysema, juvenile cirrhosis and hepatocellular carcinoma. Over 90% of clinical cases of severe α1AT deficiency are caused by the Z variant (E342K) of α1AT. The presence of the Z mutation results in misfolding and polymerization of α1AT. Due to its inherent propensity to polymerize there are no reported cases of recombinant Z α1AT production. This has created a major impediment to studying the effect of the Z mutation on α1AT. Here we report our attempts to produce recombinant Z α1AT using both Escherichia coli and Pichia pastoris as host systems. Using a range of expression vectors in E. coli we were unable to produce soluble active Z α1AT. Cytosolic expression of the Z α1AT gene in P. pastoris was successful. Monomeric and active recombinant Z α1AT was purified from the yeast cytosol using affinity chromatography and anion exchange chromatography. Biochemical analyses demonstrated that the recombinant Z α1AT has identical properties to its native counterpart purified from plasma of patients homozygous for the Z allele. A recombinant source of pathological Z α1AT will increase the chances of elucidating the mechanism of its polymerization and thus the development of therapeutic strategies. [Copyright &y& Elsevier]

In mammalian cells, multiprotein complexes form at specific genomic regulatory elements (REs) to control gene expression, which in turn is ultimately responsible for cellular identity. Consequently, insight into the molecular composition of these regulatory complexes is of major importance for our understanding of any physiological or pathological cellular state or transition. However, it remains extremely difficult to identify the protein complex(es) assembled at a specific RE in the mammalian genome using conventional approaches. We therefore developed a novel single locus isolation technique based on Transcription Activator-Like Effector (TALE) proteins termed TALE-mediated isolation of nuclear chromatin (TINC). When coupled with high-resolution mass spectrometry, TINC enables the identification and characterization of protein complexes formed at any RE of interest. Using the Nanog promoter in mouse embryonic stem cells as proof of concept, this chapter describes in detail the novel TINC methodology as well as subsequent mass spectrometric considerations., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The serpinopathies encompass a large number of diseases caused by inappropriate conformational change and self-association (polymerization) of a serpin (serine proteinase inhibitor) molecule. The most common serpinopathy is alpha(1)-antitrypsin (alpha(1)AT) deficiency, which is associated with an increased risk for liver cirrhosis, hepatocellular carcinoma and early-onset emphysema. The Z variant of alpha(1)AT, which accounts for 95% of all cases of alpha(1)AT deficiency, polymerizes during synthesis and after secretion. Here, we show using intrinsic and extrinsic fluorescence probes that Z alpha(1)AT exists in a non-native conformation. We examined the thermodynamic stability by transverse urea gradient gel electrophoresis, thermal denaturation and equilibrium guanidine hydrochloride unfolding and found that, despite structural differences between the two proteins, wild-type alpha(1)AT and Z alpha(1)AT display similar unfolding pathways and thermodynamic stabilities. Far-UV circular dichroism and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt) fluorescence suggest that the intermediate ensembles formed during unfolding of wild-type alpha(1)AT and Z alpha(1)AT are characterized by similar structural features. Kinetic analysis of the unfolding transition showed that Z alpha(1)AT unfolds at least 1.5-fold faster than the wild type. The biological implications of these data are discussed., (2009 Elsevier Ltd. All rights reserved.)

Alpha(1)-antitrypsin (alpha(1)AT), the most abundant proteinase inhibitor circulating in the blood, protects extracellular matrix proteins of the lung against proteolytic destruction by neutrophil elastase. alpha(1)AT deficiency predisposes patients to emphysema, juvenile cirrhosis and hepatocellular carcinoma. Over 90% of clinical cases of severe alpha(1)AT deficiency are caused by the Z variant (E342K) of alpha(1)AT. The presence of the Z mutation results in misfolding and polymerization of alpha(1)AT. Due to its inherent propensity to polymerize there are no reported cases of recombinant Z alpha(1)AT production. This has created a major impediment to studying the effect of the Z mutation on alpha(1)AT. Here we report our attempts to produce recombinant Z alpha(1)AT using both Escherichia coli and Pichia pastoris as host systems. Using a range of expression vectors in E. coli we were unable to produce soluble active Z alpha(1)AT. Cytosolic expression of the Z alpha(1)AT gene in P. pastoris was successful. Monomeric and active recombinant Z alpha(1)AT was purified from the yeast cytosol using affinity chromatography and anion exchange chromatography. Biochemical analyses demonstrated that the recombinant Z alpha(1)AT has identical properties to its native counterpart purified from plasma of patients homozygous for the Z allele. A recombinant source of pathological Z alpha(1)AT will increase the chances of elucidating the mechanism of its polymerization and thus the development of therapeutic strategies.

Protein aggregation is the cause of several human diseases. Understanding the molecular mechanisms involved in protein aggregation requires knowledge of the kinetics and structures populated during the reaction. Arguably, the best structurally characterized misfolding reaction is that of alpha(1)-antitrypsin. Alpha(1)-antitrypsin misfolding leads to both liver disease and emphysema and affect approximately 1 in 2000 of the population. This review will focus on the mechanism of alpha(1)-antitrypsin misfolding and the development of potential therapeutic strategies.