In the classical form of alpha1-antitrypsin (AT) deficiency, a point mutation in AT alters the folding of a liver-derived secretory glycoprotein and renders it aggregation-prone. In addition to decreased serum concentrations of AT, the disorder is characterized by accumulation of the mutant alpha1-antitrypsin Z (ATZ) variant inside cells, causing hepatic fibrosis and/or carcinogenesis by a gain-of-toxic function mechanism. The proteasomal and autophagic pathways are known to mediate degradation of ATZ. Here we show that the autophagy-enhancing drug carbamazepine (CBZ) decreased the hepatic load of ATZ and hepatic fibrosis in a mouse model of AT deficiency-associated liver disease. These results provide a basis for testing CBZ, which has an extensive clinical safety profile, in patients with AT deficiency and also provide a proof of principle for therapeutic use of autophagy enhancers.

J. Neurochem. (2012) 120 (Suppl. 1), 167–185. Abstract The amyloid cascade hypothesis of Alzheimer’s disease envisages that the initial elevation of amyloid β‐peptide (Aβ) levels, especially of Aβ1‐42, is the primary trigger for the neuronal cell death specific to onset of Alzheimer’s disease. There is now substantial evidence that brain amyloid levels are manipulable because of a dynamic equilibrium between their synthesis from the amyloid precursor protein and their removal by amyloid‐degrading enzymes (ADEs) providing a potential therapeutic strategy. Since the initial reports over a decade ago that two zinc metallopeptidases, insulin‐degrading enzyme and neprilysin (NEP), contributed to amyloid degradation in the brain, there is now an embarras de richesses in relation to this category of enzymes, which currently number almost 20. These now include serine and cysteine proteinases, as well as numerous zinc peptidases. The experimental validation for each of these enzymes, and which to target, varies enormously but up‐regulation of several of them individually in mouse models of Alzheimer’s disease has proved effective in amyloid and plaque clearance, as well as cognitive enhancement. The relative status of each of these enzymes will be critically evaluated. NEP and its homologues, as well as insulin‐degrading enzyme, remain as principal ADEs and recently discovered mechanisms of epigenetic regulation of NEP expression potentially open new avenues in manipulation of AD‐related genes, including ADEs.

Amyloidogenic processing of the amyloid precursor protein (APP) by ␤-and ␥-secretases generates several biologically active products, including amyloid-␤ (A␤) and the APP intracellular domain (AICD). AICD regulates transcription of several neuronal genes, especially the A␤-degrading enzyme, neprilysin (NEP). APP exists in several alternatively spliced isoforms, APP 695 , APP 751 , and APP 770 . We have examined whether each isoform can contribute to AICD generation and hence up-regulation of NEP expression. Using SH-SY5Y neuronal cells stably expressing each of the APP isoforms, we observed that only APP 695 up-regulated nuclear AICD levels (9-fold) and NEP expression (6-fold). A characteristic feature of Alzheimer disease (AD) 5 is the presence in the brain of extracellular amyloid plaques composed of the amyloid ␤-peptide (principally A␤ 1-40 and A␤ 1-42 ), which is derived from the transmembrane amyloid precursor protein (APP). Hence, for almost two decades, the amyloid cascade hypothesis (1, 2) has driven much AD research with a focus on the prevention of A␤ accumulation or the enhancement of its clearance as primary therapeutic strategies. In the amyloidogenic pathway of APP metabolism, A␤ is formed through the sequential actions of ␤-and ␥-secretases, whereas the non-amyloidogenic ␣-secretase pathway precludes A␤ formation. Enzymic clearance of A␤ is mediated by several enzymes, of which the metallopeptidase neprilysin (NEP) is a key contributor, and up-regulation of A␤-degrading enzymes is a potential therapeutic strategy (3, 4).Three major isoforms of APP are produced due to the alternative splicing of exons 7 and 8, which encode a 56-amino acid Kunitz-type proteinase inhibitor (KPI) domain and a 19-amino acid domain that shares sequence identity with the OX-2 antigen of thymus-derived lymphoid cells, respectively (5). The longest isoform, APP 770 , contains both the KPI and the OX-2 domains, whereas APP 751 contains only the KPI domain. The shortest isoform, APP 695 , lacks both domains. In the brain, APP 695 is expressed at high levels, and the APP 751/770 isoforms are expressed at significantly lower levels, although there are regional differences, and it has been suggested that the balance between the KPI-and non-KPI-containing isoforms may be an important factor influencing A␤ deposition (6). In the AD brain (7-9) and in response to N-methyl-Daspartate (NMDA) receptor stimulation (10, 11), there is an increase in the proportion of KPI-to non-KPI-containing isoforms of APP. This has led to the suggestion that the KPIcontaining isoforms of APP can exert important neuroprotective functions, and thus their up-regulation in the AD brain or in response to excitotoxic insult may be to protect against further neuronal loss (12, 13).A major unmet scientific need in the AD field is still to understand the normal function of APP (14). An added complexity is whether the different APP isoforms have similar or distinct localizations, metabolism, and roles (15). A long standing enigma in APP biology has additi...

Unlike other cytochromes, c‐type cytochromes have two covalent bonds formed between the two vinyl groups of haem and two cysteines of the protein. This haem ligation requires specific assembly proteins in prokaryotes or eukaryotic mitochondria and chloroplasts. Here, it is shown that Bordetella pertussis is an excellent bacterial model for the widespread system II cytochrome c synthesis pathway. Mutations in four different genes (ccsA, ccsB, ccsX and dipZ) result in B. pertussis strains unable to synthesize any of at least seven c‐type cytochromes. Using a cytochrome c4:alkaline phosphatase fusion protein as a bifunctional reporter, it was demonstrated that the B. pertussis wild‐type and mutant strains secrete an active alkaline phosphatase fusion protein. However, unlike the wild type, all four mutants are unable to attach haem covalently, resulting in a degraded N‐terminal apocytochrome c4 component. Thus, apocytochrome c secretion is normal in each of the four mutants, but all are defective in a periplasmic assembly step (or export of haem). CcsX is related to thioredoxins, which possess a conserved CysXxxXxxCys motif. Using phoA gene fusions as reporters, CcsX was proven to be a periplasmic thioredoxin‐like protein. Both the B. pertussis dipZ (i.e. dsbD) and ccsX mutants are corrected for their assembly defects by the thiol‐reducing compounds, dithiothreitol and 2‐mercaptoethanesulphonic acid. These results indicate that DipZ and CcsX are required for the periplasmic reduction of the cysteines of apocytochromes c before ligation. In contrast, the ccsA and ccsB mutants are not corrected by exogenous reducing agents, suggesting that CcsA and CcsB are required for the haem ligation step itself in the periplasm (or export of haem to the periplasm). Related to this suggestion, the topology of CcsB was determined experimentally, demonstrating that CcsB has four transmembrane domains and a large 435‐amino‐acid periplasmic region.

In the classical form of ␣ 1 -antitrypsin deficiency, a mutant protein accumulates in a polymerized form in the endoplasmic reticulum (ER) of liver cells causing liver damage and carcinogenesis by a gain-of-toxic function mechanism. Recent studies have indicated that the accumulation of mutant ␣ 1 -antitrypsin Z in the ER specifically activates the autophagic response but not the unfolded protein response and that autophagy plays a critical role in disposal of insoluble ␣ 1 -antitrypsin Z. In this study, we used genomic analysis of the liver in a novel transgenic mouse model with inducible expression to screen for changes in gene expression that would potentially define how the liver responds to accumulation of this mutant protein. There was no unfolded protein response. Of several distinct gene expression profiles, marked up-regulation of regulator of G signaling (RGS16) was particularly notable. RGS16 did not increase when model systems were exposed to classical inducers of ER stress, including tunicamycin and calcium ionophore, or when a nonpolymerogenic ␣ 1 -antitrypsin mutant accumulated in the ER. RGS16 was up-regulated in livers from patients with ␣ 1 -antitrypsin deficiency, and the degree of up-regulation correlated with the hepatic levels of insoluble ␣ 1 -antitrypsin Z protein. Taken together, these results indicate that expression of RGS16 is an excellent marker for the distinct form of "ER stress" that occurs in ␣ 1 -antitrypsin deficiency, presumably determined by the aggregation-prone properties of the mutant protein that characterizes the deficiency.The histological hallmark of the classical form of ␣ 1 -antitrypsin (AT) 2 deficiency is liver cells containing periodic acidSchiffϩ/diastase-resistant globules. From many years of research on the disease, we now know that these globules represent rough endoplasmic reticulum (ER) distended by accumulation of the mutant ATZ molecule (where ATZ is the Z variant of ␣ 1 -antitrypsin). The wild type AT is a classical liverderived secretory glycoprotein that is delivered by the circulating blood to tissues to subserve its predominant function of inhibiting the neutrophil serine proteases neutrophil elastase, cathepsin G, and proteinase 3. The point mutation that characterizes the ATZ variant converts glutamate 342 to lysine and is sufficient to result in selective retention of the glycoprotein in the ER (reviewed in Refs. 1, 2). Thus, AT deficiency could be considered a prototype, naturally occurring "ER stress" state. Characterization of the structure of AT and its functional correlates led to the remarkable observation that the substitution of lysine for glutamate 342 conferred on the ATZ molecule a tendency to polymerize and aggregate (3, 4). Although it is still not clear whether the tendency to polymerize is the cause, or an effect, of ER retention, there is clear-cut evidence that polymers and aggregates of this molecule are formed in the ER, and there is growing evidence that these polymers and aggregates play a role in how liver cells respond and wh...