Alpha 1 -antitrypsin is the most abundant circulating protease inhibitor. The severe Z deficiency allele (Glu342Lys) causes the protein to undergo a conformational transition and form ordered polymers that are retained within hepatocytes. This causes neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. We have developed a conformation-specific monoclonal antibody (2C1) that recognizes the pathological polymers formed by a 1 -antitrypsin. This antibody was used to characterize the Z variant and a novel shutter domain mutant (His334Asp; a 1 -antitrypsin King's) identified in a 6-week-old boy who presented with prolonged jaundice. His334Asp a 1 -antitrypsin rapidly forms polymers that accumulate within the endoplasmic reticulum and show delayed secretion when compared to the wild-type M a 1 -antitrypsin. The 2C1 antibody recognizes polymers formed by Z and His334Asp a 1 -antitrypsin despite the mutations directing their effects on different parts of the protein. This antibody also recognized polymers formed by the Siiyama (Ser53Phe) and Brescia (Gly225Arg) mutants, which also mediate their effects on the shutter region of a 1 -antitrypsin. Conclusion: Z and shutter domain mutants of a 1 -antitrypsin form polymers with a shared epitope and so are likely to have a similar structure. (HEPATOLOGY 2010;52:1078-1088 T he serpinopathies are conformational diseases characterized by the polymerization and intracellular retention of members of the serine protease inhibitor or serpin superfamily of proteins.1 The best known is a 1 -antitrypsin deficiency, with the most common severe deficiency allele being the Z mutation (Glu342Lys). This mutation results in the retention of ordered polymers of a 1 -antitrypsin as periodic acid Schiff positive inclusion bodies within the endoplasmic reticulum (ER) of hepatocytes.2 These inclusions predispose the individual homozygous for the Z variant of the a 1 -antitrypsin protease inhibitor (PI*Z) to neonatal hepatitis, cirrhosis, and rarely, hepatocellular carcinoma.3 Deficiency of circulating a 1 -antitrypsin results in early onset panlobular emphysema.

The serpinopathies result from the ordered polymerization of mutants of members of the serine proteinase inhibitor (serpin) superfamily. These polymers are retained within the cell of synthesis where they cause a toxic gain of function. The serpinopathies are exemplified by inclusions that form with the common severe Z mutant of α 1 -antitrypsin that are associated with liver cirrhosis. There is considerable controversy as to the pathway of serpin polymerization and the structure of pathogenic polymers that cause disease. We have used synthetic peptides, limited proteolysis, monoclonal antibodies, and ion mobility-mass spectrometry to characterize the polymerogenic intermediate and pathological polymers formed by Z α 1 -antitrypsin. Our data are best explained by a model in which polymers form through a single intermediate and with a reactive center loop-β-sheet A linkage. Our data are not compatible with the recent model in which polymers are linked by a β-hairpin of the reactive center loop and strand 5A. Understanding the structure of the serpin polymer is essential for rational drug design strategies that aim to block polymerization and so treat α 1 -antitrypsin deficiency and the serpinopathies.alpha-1-antitrypsin deficiency | protein folding | serpins | polymers | cirrhosis

The severe, early onset emphysema that occurs in patients with circulating deficiency of a 1 -antitrypsin (a 1 -AT) attests to the importance of this protease inhibitor in maintaining lung parenchymal integrity. It has led to the powerful concept of protease:antiprotease balance being crucial to alveolar homeostasis.Pathogenic mutations cause a 1 -AT to self-associate into polymer chains that accumulate intracellularly rather than proceeding along the secretory pathway. Polymerisation of a 1 -AT abolishes antiprotease activity and confers toxic gain-of-function effects. Since a 1 -AT is predominantly synthesised in the liver, where it does not play a major homeostatic role, the directly toxic effects of polymerisation are clearest here. However, data from molecular, cellular, animal and ex vivo studies indicate that intrapulmonary polymerisation of a 1 -AT and inflammatory positive feedback loops may augment the destructive effects of decreased antiprotease levels in the lung.This review integrates the findings from these different approaches and highlights how multiple pathways may converge to give the severe, panacinar emphysema phenotype seen in a 1 -AT deficiency.

Summary Background Whole-body magnetic resonance imaging (WB-MRI) could be an alternative to multi-modality staging of non-small-cell lung cancer (NSCLC), but its diagnostic accuracy, effect on staging times, number of tests needed, cost, and effect on treatment decisions are unknown. We aimed to prospectively compare the diagnostic accuracy and efficiency of WB-MRI-based staging pathways with standard pathways in NSCLC. Methods The Streamline L trial was a prospective, multicentre trial done in 16 hospitals in England. Eligible patients were 18 years or older, with newly diagnosed NSCLC that was potentially radically treatable on diagnostic chest CT (defined as stage IIIb or less). Exclusion criteria were severe systemic disease, pregnancy, contraindications to MRI, or histologies other than NSCLC. Patients underwent WB-MRI, the result of which was withheld until standard staging investigations were complete and the first treatment decision made. The multidisciplinary team recorded its treatment decision based on standard investigations, then on the WB-MRI staging pathway (WB-MRI plus additional tests generated), and finally on all tests. The primary outcome was difference in per-patient sensitivity for metastases between standard and WB-MRI staging pathways against a consensus reference standard at 12 months, in the per-protocol population. Secondary outcomes were difference in per-patient specificity for metastatic disease detection between standard and WB-MRI staging pathways, differences in treatment decisions, staging efficiency (time taken, test number, and costs) and per-organ sensitivity and specificity for metastases and per-patient agreement for local T and N stage. This trial is registered with the International Standard Randomised Controlled Trial registry, number ISRCTN50436483, and is complete. Findings Between Feb 26, 2013, and Sept 5, 2016, 976 patients were screened for eligibility. 353 patients were recruited, 187 of whom completed the trial; 52 (28%) had metastasis at baseline. Pathway sensitivity was 50% (95% CI 37–63) for WB-MRI and 54% (41–67) for standard pathways, a difference of 4% (−7 to 15, p=0·73). No adverse events related to imaging were reported. Specificity did not differ between WB-MRI (93% [88–96]) and standard pathways (95% [91–98], p=0·45). Agreement with the multidisciplinary team's final treatment decision was 98% for WB-MRI and 99% for the standard pathway. Time to complete staging was shorter for WB-MRI (13 days [12–14]) than for the standard pathway (19 days [17–21]); a 6-day (4–8) difference. The number of tests required was similar WB-MRI (one [1–1]) and standard pathways (one [1–2]). Mean per-patient costs were £317 (273–361) for WBI-MRI and £620 (574–666) for standard pathways. Interpretation WB-MRI staging pathways have similar accuracy to standard pathways, and reduce the staging time and costs. Funding UK National Institute...

alpha1-Antitrypsin is the prototypical member of the serine proteinase inhibitor or serpin superfamily of proteins. The family includes alpha1-antichymotrypsin, C1 inhibitor, antithrombin and neuroserpin, which are all linked by a common molecular structure and the same suicidal mechanism for inhibiting their target enzymes. Point mutations result in an aberrant conformational transition and the formation of polymers that are retained within the cell of synthesis. The intracellular accumulation of polymers of mutant alpha1-antitrypsin and neuroserpin results in a toxic gain-of-function phenotype associated with cirrhosis and dementia respectively. The lack of important inhibitors results in overactivity of proteolytic cascades and diseases such as COPD (chronic obstructive pulmonary disease) (alpha1-antitrypsin and alpha1-antichymotrypsin), thrombosis (antithrombin) and angio-oedema (C1 inhibitor). We have grouped these conditions that share the same underlying disease mechanism together as the serpinopathies. In the present review, the molecular and pathophysiological basis of alpha1-antitrypsin deficiency and other serpinopathies are considered, and we show how understanding this unusual mechanism of disease has resulted in the development of novel therapeutic strategies.

Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.—Ordóñez, A., Pérez, J., Tan, L., Dickens, J. A., Motamedi-Shad, N., Irving, J. A., Haq, I., Ekeowa, U., Marciniak, S. J., Miranda, E., Lomas, D. A. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

Background Whole-body MRI (WB-MRI) could be an alternative to multimodality staging of colorectal cancer, but its diagnostic accuracy, effect on staging times, number of tests needed, cost, and effect on treatment decisions are unknown. We aimed to prospectively compare the diagnostic accuracy and efficiency of WB-MRI-based staging pathways with standard pathways in colorectal cancer. Methods The Streamline C trial was a prospective, multicentre trial done in 16 hospitals in England. Eligible patients were 18 years or older, with newly diagnosed colorectal cancer. Exclusion criteria were severe systemic disease, pregnancy, contraindications to MRI, or polyp cancer. Patients underwent WB-MRI, the result of which was withheld until standard staging investigations were complete and the first treatment decision made. The multidisciplinary team recorded its treatment decision based on standard investigations, then on the WB-MRI staging pathway (WB-MRI plus additional tests generated), and finally on all tests. The primary outcome was difference in per-patient sensitivity for metastases between standard and WB-MRI staging pathways against a consensus reference standard at 12 months, in the per-protocol population. Secondary outcomes were difference in per-patient specificity for metastatic disease detection between standard and WB-MRI staging pathways, differences in treatment decisions, staging efficiency (time taken, test number, and costs), and per-organ sensitivity and specificity for metastases and per-patient agreement for local T and N stage. This trial is registered with the International Standard Randomised Controlled Trial registry, number ISRCTN43958015, and is complete.

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