Engineering the serpin α1 -antitrypsin: A diversity of goals and techniques.
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ABSTRACT:
SUBMITTER: Scott BM
PROVIDER: S-EPMC7096715 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Protein glycosylation commonly stabilizes proteins thereby increasing protein half-lives and protecting against denaturation or proteolytic degradation. While generally beneficial, such stabilization is potentially disadvantageous in the case of inhibitory serpins. These protease inhibitors are metastable and a conformational transition to a more stable form is key to their function. Instability is therefore essential for these inhibitory serpins and mutagenesis has demonstrated that substantial stabilization results in compromised function. We have used optical spectroscopy and hydrogen/deuterium exchange and mass spectrometry to investigate the effects of glycosylation on the human serpin alpha-1 antitrypsin (α(1)-AT). Previous studies found that unglycosylated recombinant α(1)-AT populates a molten globule at low denaturant and that the ability to populate this state is correlated with efficient protease inhibition. Further, a high degree of conformational flexibility was found in several important regions. Guanidine hydrochloride denaturation monitored by circular dichroism indicates that plasma α(1)-AT, which is glycosylated at 3 sites, is substantially stabilized relative to the unglycosylated form. However, hydrogen exchange reveals complete loss of protection in plasma α(1)-AT above 1 M GuHCl, similar to what is seen for the recombinant form. Sugars therefore appear to stabilize the compact denatured state of α(1)-AT without significant stabilization of the folded state. Native state hydrogen exchange reveals minor perturbations to native flexibility, but high flexibility in key regions such as the f helix is conserved. β-strand 1c is stabilized in plasma α(1)-AT, which may confer increased resistance to forming pathogenic polymers. Overall, our results indicate that glycosylation of inhibitory serpins does not interfere with either native state flexibility or the native instability that is required for efficient function, though it may confer resistance to degradation by proteases and thus extend the half-life of circulating serpins.
| S-EPMC3134971 | biostudies-literature
Project description:The misfolding of serpins is linked to several genetic disorders including emphysema, thrombosis, and dementia. During folding, inhibitory serpins are kinetically trapped in a metastable state in which a stretch of residues near the C terminus of the molecule are exposed to solvent as a flexible loop (the reactive center loop). When they inhibit target proteases, serpins transition to a stable state in which the reactive center loop forms part of a six-stranded ?-sheet. Here, we use hydrogen-deuterium exchange mass spectrometry to monitor region-specific folding of the canonical serpin human ?(1)-antitrypsin (?(1)-AT). We find large differences in the folding kinetics of different regions. A key region in the metastable ? stable transition, ?-strand 5A, shows a lag phase of nearly 350 s. In contrast, the "B-C barrel" region shows no lag phase and the incorporation of the C-terminal residues into ?-sheets B and C is largely complete before the center of ?-sheet A begins to fold. We propose this as the mechanism for trapping ?(1)-AT in a metastable form. Additionally, this separation of timescales in the folding of different regions suggests a mechanism by which ?(1)-AT avoids polymerization during folding.
| S-EPMC3311335 | biostudies-other
Project description:Genetic mutations predispose the serine protease inhibitor α1-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 α1-antitrypsin variants in solution. Here we apply NMR spectroscopy to patient-derived samples of α1-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). By comparison with perturbations induced by binding of a small-molecule inhibitor of misfolding we conclude that they arise from rapid exchange between the native conformation and a well-populated intermediate state. The observation that this intermediate is stabilised by inhibitor binding suggests a paradoxical approach to the targeted treatment of protein misfolding disorders, wherein the stabilisation of disease-associated states provides selectivity while inhibiting further transitions along misfolding pathways.
| S-EPMC7732992 | biostudies-literature
Project description:The aggregation of antitrypsin into polymers is one of the causes of neonatal hepatitis, cirrhosis, and emphysema. A similar reaction resulting in disease can occur in other human serpins, and collectively they are known as the serpinopathies. One possible therapeutic strategy involves inhibiting the conformational changes involved in antitrypsin aggregation. The citrate ion has previously been shown to prevent antitrypsin aggregation and maintain the protein in an active conformation; its mechanism of action, however, is unknown. Here we demonstrate that the citrate ion prevents the initial misfolding of the native state to a polymerogenic intermediate in a concentration-dependent manner. Furthermore, we have solved the crystal structure of citrate bound to antitrypsin and show that a single citrate molecule binds in a pocket between the A and B beta-sheets, a region known to be important in maintaining antitrypsin stability.
| S-EPMC2590919 | biostudies-literature
Project description:Clinical benefit of Alpha1-Proteinase Inhibitor (Human) (A1-PI) products for Alpha1-antitrypsin deficiency (AATD) is uncertain, based on a systematic review of observational studies and randomized controlled trials (RCTs) in AATD of Alpha1-Proteinase Inhibitor (Human) (A1-PI) products. At the recommended dose, A1-PI products raise its serum concentration but do not normalize levels. Observational studies suggest A1-PI might modestly slow progression of airflow limitation in patients with intermediate airflow obstruction, a finding not confirmed by three placebo-controlled RCTs of limited power, which showed non-significant rates of forced expiratory volume in 1 s (FEV1) change favoring placebo. These RCTs found trends favoring A1-PI in loss of high-resolution computerized tomographic (HRCT) lung density. While two meta-analyses of HRCT lung density change in RCTs achieved significance favoring A1-PI arms, clinical benefit remains uncertain. HRCT lung density measurements don't distinguish changes in measured density due to fluid shifts into and out of the lungs and changes in lung inflammation from those due to progressive loss of alveolar mass. A meta-analysis of RCTs found exacerbations significantly increased in A1-PI groups compared to placebo. No RCTs have shown favorable effects of A1-PI on mortality, FEV1, 6-min walking distance, quality of life, change in diffusion capacity of carbon monoxide (DLCO), or exacerbation frequency. A fourth RCT comparing two dose regimens of A1-PI is underway. RCTs have not provided evidence of clinical benefit in terms of how patients feel, function, or survive. Results have implications for the design of future clinical trials of A1-PI and potentially other products targeting AATD-associated emphysema.
| S-EPMC11190464 | biostudies-literature
Project description:Corticosteroid resistance after acute graft-versus-host disease (SR-aGVHD) results in high morbidity and mortality after allogeneic hematopoietic cell transplantation. Current immunosuppressive therapies for SR-aGVHD provide marginal effectiveness because of poor response or excessive toxicity, primarily from infection. α1-Antitrypsin (AAT), a naturally abundant serine protease inhibitor, is capable of suppressing experimental GVHD by downmodulating inflammation and increasing ratios of regulatory (Treg) to effector T cells (Teffs). In this prospective multicenter clinical study, we sought to determine the safety and response rate of AAT administration in SR-aGVHD. Forty patients with a median age of 59 years received intravenous AAT twice weekly for 4 weeks as first-line treatment of SR-aGVHD. The primary end point was overall response rate (ORR), the proportion of patients with SR-aGVHD in complete (CR) or partial response by day 28 without addition of further immunosuppression. Treatment was well tolerated without drug-related adverse events. A significant increase in serum levels of AAT was observed after treatment. The ORR and CR rates by day 28 were 65% and 35%, respectively, and included responses in all aGVHD target organs. At day 60, responses were sustained in 73% of patients without intervening immunosuppression. Infectious mortality was 10% at 6 months and 2.5% within 30 days of last AAT infusion. Consistent with preclinical data, correlative samples showed an increase in ratio of activated Tregs to Teffs after AAT treatment. These data suggest that AAT is safe and may be potentially efficacious in treating SR-aGVHD. This trial was registered at www.clinicaltrials.gov as #NCT01700036.
| S-EPMC5865235 | biostudies-literature
Project description:BACKGROUND:Alpha-1-antitrypsin (AAT) deficiency (AATD) of Z, Mmalton, Siiyama type is associated with liver storage of the mutant proteins and liver disease. The Z variant can be diagnosed on isoelectric focusing (IEF) while Mmalton and Siiyama may be missed or misdiagnosed with this technique. Therefore, molecular analysis is mandatory for their characterization. In particular, that holds true for the Mmalton variant as on IEF profile it resembles the wild M2 subtype. METHODS:This is a retrospective analysis involving review of medical records and of liver biopsy specimens from a series of Mmalton, Z and Siiyama Alpha-1-antitrypsin deficiency patients. The review has been implemented by additional histological stains, electron microscopic observations and 3-D modeling studies of the sites of the mutations. RESULTS:Z, Mmalton and Siiyama liver specimen contained characteristic intrahepatocytic PAS-D globules. The globules differed in the three variants as only Mmalton cases showed dark basophilic precipitates within the AAT inclusions. The precipitates were visualized in haematoxylin-eosin (H.E.) stained preparations and corresponded to calcium precipitates as demonstrated by von Kossa staining. On immunohistochemistry, ZAAT inclusions were stained by polyclonal as well as monoclonal noncommercial anti-AAT antibody (AZT11), whilst Mmalton and Siiyama inclusion bodies remained negative with the monoclonal anti-Z antibody. 3-D protein analysis allowed to predict more severe misfolding of the Mmalton molecule as compared to Z and Siiyama that could trigger anomalous interaction with endoplasmic reticulum chaperon proteins, namely calcium binding proteins. CONCLUSIONS:Mmalton AAT inclusion bodies contain calcium precipitates inside them that allow the differential diagnosis with Siiyama and ZAAT inclusions in routine histological sections. The study has confirmed the specificity of the monoclonal AZT11 for the Z mutant. Thus, the combination of these two features is crucial for the distinction between the three variants and for predicting the genotype, whose confirmation would definitely require molecular analysis. Our study provides new data on the pathomorphogenesis of Mmalton inclusion bodies whose mineralization could play a central role in disease pathogenesis of Mmalton that is distinct from the Z and Siiyama variants. Calcium is known to be a major effector of cell death either via the increased intracellular concentration or the alteration of homeostasis.
| S-EPMC5956786 | biostudies-literature
Project description:Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder which most commonly manifests as pulmonary emphysema. Accordingly, alpha-1 antitrypsin (AAT) augmentation therapy aims to reduce the progression of emphysema, as achieved by life-long weekly slow-drip infusions of plasma-derived affinity-purified human AAT. However, not all AATD patients will receive this therapy, due to either lack of medical coverage or low patient compliance. To circumvent these limitations, attempts are being made to develop lung-directed therapies, including inhaled AAT and locally-delivered AAT gene therapy. Lung transplantation is also an ultimate therapy option. Although less common, AATD patients also present with disease manifestations that extend beyond the lung, including vasculitis, diabetes and panniculitis, and appear to experience longer and more frequent hospitalization times and more frequent pneumonia bouts. In the past decade, new mechanism-based clinical indications for AAT therapy have surfaced, depicting a safe, anti-inflammatory, immunomodulatory and tissue-protective agent. Introduced to non-AATD individuals, AAT appears to provide relief from steroid-refractory graft-versus-host disease, from bacterial infections in cystic fibrosis and from autoimmune diabetes; preclinical studies show benefit also in multiple sclerosis, ulcerative colitis, rheumatoid arthritis, acute myocardial infarction and stroke, as well as ischemia-reperfusion injury and aberrant wound healing processes. While the current augmentation therapy is targeted towards treatment of emphysema, it is suggested that AATD patients may benefit from AAT augmentation therapy geared towards extrapulmonary pathologies as well. Thus, development of mechanism-based, context-specific AAT augmentation therapy protocols is encouraged. In the current review, we will discuss extrapulmonary manifestations of AATD and the potential of AAT augmentation therapy for these conditions.
| S-EPMC6361480 | biostudies-literature
Project description:Alpha(1)-antitrypsin is a 45-kDa (394-residue) serine protease inhibitor synthesized by hepatocytes, which is released into the circulatory system and protects the lung from the actions of neutrophil elastase via a conformational transition within a dynamic inhibitory mechanism. Relatively common point mutations subvert this transition, causing polymerisation of ?(1)-antitrypsin and deficiency of the circulating protein, predisposing carriers to severe lung and liver disease. We have assigned the backbone resonances of ?(1)-antitrypsin using multidimensional heteronuclear NMR spectroscopy. These assignments provide the starting point for a detailed solution state characterization of the structural properties of this highly dynamic protein via NMR methods.
| S-EPMC3438405 | biostudies-literature
Project description:Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder associated to early onset emphysema, mainly imputable to Pi*ZZ genotype. In spite of the serious potential effects, many AATD individuals do not develop emphysema. To identify genes/variants potentially involved in emphysema development we studied 4 AATD families. Each family had at least one affected sibling with emphysema and one non-affected. Whole Exome Sequencing (WES) was performed on genomic DNA isolated from 9 individuals with AATD (4 affected/5 non-affected). Genetic variants confirmed at least in three families were prioritized using QueryOR and network analysis was used to verify enriched pathways. In affected subjects: 14 genes (57% immune-related) segregated in a recessive model and 21 (29% immune-related) in a dominant model. In non-affected subjects: 21 genes (43% immune-related) segregated in a recessive model and 50 (24% immune-related) in a dominant model. In affected siblings immune genes had an activating function, while where immune-suppressing in non-affected siblings involving antigen processing, MHC-I presentation, TCR and PD-1 signalling. This study describes possible genetic susceptibility factors for emphysema development in AATD, and suggests that gene variants involved in regulation of immune homeostasis and maintenance of self-tolerance contribute to the development or suppression of the disease.
| S-EPMC6739380 | biostudies-literature