Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

Monash University

A 191-kb human bacterial artificial chromosome (BAC) containing the human alpha-globin genomic locus was used to generate transgenic mice that express, exclusively, human alpha-globin ((hu)alpha-globin). Expression of (hu)alpha-globin reaches a level of 36% of that of endogenous mouse alpha-globin ((mu)alpha-globin) on a heterozygous mouse alpha-thalassemia background ((mu)alpha-globin knockout, (mu)alpha(+/-)). Hemizygous transgenic mice carrying the (hu)alpha-globin locus on a heterozygous knockout background ((hu)alpha(+/0), (mu)alpha(++/--)) demonstrated complementation of most hematologic parameters. By crossing (hu)alpha(+/0), (mu)alpha(++/--) mice, we were able to generate mice entirely dependent on (hu)alpha-globin synthesis. Breeding and fluorescent in situ hybridization studies demonstrate that only mice homozygous for the transgene were able to rescue embryonic lethal homozygous (mu)alpha-globin knockout embryos ((mu)alpha(--/--)). Adult rescued mice produce hemoglobin at levels similar to wild-type mice, with partial red cell complementation based on mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and red cell distribution width (RDW) measurements. Significant erythrocythemia above wild-type levels seems to be the main compensatory mechanism for the normalization of the hemoglobin levels in the rescued animals. Our studies demonstrate that the (hu)alpha-globin locus in the 191-kb transgene contains all the necessary elements for the regulated expression of (hu)alpha-globin in transgenic mice. This animal model should be valuable for studying the mechanisms regulating (hu)alpha-globin production and for development of therapeutic strategies for beta-thalassemia based on downregulation of alpha-globin expression.

A 191-kb genomic fragment containing the human alpha-globin locus can rescue alpha-thalassemic mice.

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.

Serpin polymerization and its role in disease--the molecular basis of alpha1-antitrypsin deficiency.

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.

Expression, purification and characterization of recombinant Z alpha(1)-antitrypsin--the most common cause of alpha(1)-antitrypsin deficiency.

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.

Kinetic instability of the serpin Z alpha1-antitrypsin promotes aggregation.

The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α(1)-antitrypsin (α(1)AT) deficiency cases. It leads to increased polymerization of the serpin α(1)AT 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 α(1)AT and analyzed their fluorescence properties. α(1)AT 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 α(1)AT. 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 α(1)AT compared to M α(1)AT. 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.

Structural change in β-sheet A of Z α(1)-antitrypsin is responsible for accelerated polymerization and disease.

The human serine protease inhibitor (serpin) α-1 antitrypsin (α1-AT) protects tissues from proteases of inflammatory cells. The most common disease-causing mutation in α1-AT is the Z-mutation (E342K) that results in an increased propensity of α1-AT to polymerize in the ER of hepatocytes, leading to a lack of secretion into the circulation. The structural consequences of this mutation, however, remain elusive. We report a comparative molecular dynamics investigation of the native states of wild-type and Z α1-AT, revealing a striking contrast between their structures and dynamics in the breach region at the top of β-sheet A, which is closed in the wild-type simulations but open in the Z form. Our findings are consistent with experimental observations, notably the increased solvent exposure of buried residues in the breach region in Z, as well as polymerization via domain swapping, whereby the reactive center loop is rapidly inserted into an open A-sheet before proper folding of the C-terminal β-strands, allowing C-terminal domain swapping with a neighboring molecule. Taken together, our experimental and simulation data imply that mutations at residue 342 that either stabilize an open form of the top of β-sheet A or increase the local flexibility in this region, may favor polymerization and hence aggregation.

Conformational properties of the disease-causing Z variant of α1-antitrypsin revealed by theory and experiment.

Cellular injury causes a myriad of processes that affect proteostasis. We describe nucleocytoplasmic coagulation (NCC), an intracellular disulfide-dependent protein crosslinking event occurring upon late-stage cell death that orchestrates the proteolytic removal of misfolded proteins. In vitro and in vivo models of neuronal injury show that NCC involves conversion of soluble intracellular proteins, including tubulin, into insoluble oligomers. These oligomers, also seen in human brain tissue following neurotrauma, act as a cofactor and substrate for the plasminogen-activating system. In plasminogen(-/-) mice, levels of misfolded β-tubulin were elevated and its clearance delayed following neurotrauma, demonstrating a requirement for plasminogen in the removal of NCC constituents. While additional in vivo studies will further dissect this phenomenon, our study clearly shows that NCC, a process analogous to the formation of thrombi, generates an aggregated protein scaffold that limits release of cellular components and recruits clearance mechanisms to the site of injury.

Nucleocytoplasmic coagulation: an injury-induced aggregation event that disulfide crosslinks proteins and facilitates their removal by plasmin.

α(1)-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 α(1)-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 α(1)-Antitrypsin has never been examined. We have therefore created a range of α(1)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 α(1)-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.

The roles of helix I and strand 5A in the folding, function and misfolding of α1-antitrypsin.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form, however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-crosslinked state via the process of 'Nucleocytoplasmic Coagulation'. Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of Nucleocytoplasmic Coagulation. Despite the fact that disulfide-crosslinking is a prominent feature of GAPDH aggregation, our data shows that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications, within the context of the high-resolution native GAPDH structure, suggested that oxidation of methionine-46 may instigate aggregation. We confirmed this by mutating methionine-46 to leucine, which rendered GAPDH highly-resistant to free radical-induced aggregation. Molecular Dynamics simulations suggest that oxidation of methionine-46 triggers a local increase in the conformational plasticity of GAPDH, which likely promotes further oxidation and eventual aggregation. Hence, methionine-46 represents a "linchpin", whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation and disulfide-crosslinking of GAPDH. A critical role for linchpin residues in Nucleocytoplasmic Coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, since disulfide-crosslinked aggregates of GAPDH arise in many disorders, and because methionine-46 is irrelevant to native GAPDH function, mutation of methionine-46 in models of disease should allow the unequivocally assessment of whether GAPDH aggregation influences disease progression.

Anja S. Knaupp

Department of Anatomy and Developmental Biology,
Australian Regenerative Medicine Institute

Monash University

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Anja S. Knaupp

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Anatomy and Developmental Biology,Australian Regenerative Medicine Institute

Monash University

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Department of Anatomy and Developmental Biology,
Australian Regenerative Medicine Institute