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In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC ؊ strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC ؊ strain, even though the dsbA ؊ single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses.Most periplasmic Escherichia coli proteins contain at least two cysteine residues and many are stable and active only when these cysteines form their native disulfide bond pairings (1). In E. coli, a family of thioldisulfide oxidoreductases ensures that periplasmic and secreted proteins form correct disulfide bonds. DsbA is the primary disulfide oxidant in the periplasm. It rapidly donates its disulfide directly to substrate proteins and oxidizes them (2). DsbA is believed to act as a relatively nonspecific oxidant, joining any two cysteines that approach each other (3). A dsbA Ϫ strain shows several in vivo phenotypes, including attenuated virulence and loss of motility, because of the absence of disulfide bonds in proteins involved in these pathways (4, 5). DsbC, a second periplasmic thiol-disulfide oxidoreductase, appears to function as a disulfide isomerase both in vitro and in vivo. In vitro, DsbC has been shown to rearrange non-native disulfides in well studied isomerization substrates such as BPTI and RNase A (6, 7). In vivo, DsbC is required for full activity of a handful of proteins containing at least one non-consecutive disulfide bond (1). We have found that the periplasmic proteins RNase I (four disulfides, one non-consecutive) and MepA (three disulfides, two non-consecutive) require DsbC for their stability and, in the case of RNase I, in vivo activity (1). Berkmen et al. (3) recently showed that the folding of Agp (three consecutive disulfides) becomes DsbC-dependent with the introduction of a non-consecutive disulfide bond. These results s...

The Gram-negative bacterial envelope is bounded by two membranes. Disulfide bond formation and isomerization in this oxidizing environment are catalyzed by DsbA and DsbC, respectively. It remains unknown when and how the Dsb proteins participate in the biogenesis of outer membrane proteins, which are transported across the cell envelope after their synthesis. The Escherichia coli protein LptD is an integral outer membrane protein that forms an essential complex with the lipoprotein LptE. We show that oxidation of LptD is not required for the formation of the LptD/E complex but it is essential for function. Remarkably, none of the cysteines in LptD are essential because either of two nonconsecutive disulfide bonds suffices for function. Oxidation of LptD, which is efficiently catalyzed by DsbA, does not involve the isomerase DsbC, but it requires LptE. Thus, oxidation is completed only after LptD interacts with LptE, an interaction that occurs at the outer membrane and seems necessary for LptD folding.β-barrel protein | lipoprotein | protein folding

In Escherichia coli, a family of periplasmic disulfide oxidoreductases catalyzes correct disulfide bond formation in periplasmic and secreted proteins. Despite the importance of native disulfide bonds in the folding and function of many proteins, a systematic investigation of the in vivo substrates of E. coli periplasmic disulfide oxidoreductases, including the well characterized oxidase DsbA, has not yet been performed. We combined a modified osmotic shock periplasmic extract and twodimensional gel electrophoresis to identify substrates of the periplasmic oxidoreductases DsbA, DsbC, and DsbG. We found 10 cysteine-containing periplasmic proteins that are substrates of the disulfide oxidase DsbA, including PhoA and FlgI, previously established DsbA substrates. This technique did not detect any in vivo substrates of DsbG, but did identify two substrates of DsbC, RNase I and MepA. We confirmed that RNase I is a substrate of DsbC both in vivo and in vitro. This is the first time that DsbC has been shown to affect the in vivo function of a native E. coli protein, and the results strongly suggest that DsbC acts as a disulfide isomerase in vivo. We also demonstrate that DsbC, but not DsbG, is critical for the in vivo activity of RNase I, indicating that DsbC and DsbG do not function identically in vivo. The absence of substrates for DsbG suggests either that the in vivo substrate specificity of DsbG is more limited than that of DsbC or that DsbG is not active under the growth conditions tested. Our work represents one of the first times the in vivo substrate specificity of a folding catalyst system has been systematically investigated. Because our methodology is based on the simple assumption that the absence of a folding catalyst should cause its substrates to be present at decreased steady-state levels, this technique should be useful in analyzing the substrate specificity of any folding catalyst or chaperone for which mutations are available.The Escherichia coli periplasm contains a family of disulfide bond-forming (Dsb) proteins that catalyze disulfide bond formation and rearrangement. This family of thiol-disulfide oxidoreductases can be divided into two pathways: an oxidation pathway, consisting of the inner membrane protein DsbB and the periplasmic protein DsbA, and an isomerization pathway,

Plasma biomarkers related to amyloid, tau, and neurodegeneration (ATN) show great promise for identifying these pathological features of Alzheimer’s Disease (AD) as shown by recent clinical studies and selected autopsy studies. We have evaluated ATN plasma biomarkers in a series of 312 well-characterized longitudinally followed research subjects with plasma available within 5 years or less before autopsy and examined these biomarkers in relation to a spectrum of AD and related pathologies. Plasma Aβ42, Aβ40, total Tau, P-tau181, P-tau231 and neurofilament light (NfL) were measured using Single molecule array (Simoa) assays. Neuropathological findings were assessed using standard research protocols. Comparing plasma biomarkers with pathology diagnoses and ratings, we found that P-tau181 (AUC = 0.856) and P-tau231 (AUC = 0.773) showed the strongest overall sensitivity and specificity for AD neuropathological change (ADNC). Plasma P-tau231 showed increases at earlier ADNC stages than other biomarkers. Plasma Aβ42/40 was decreased in relation to amyloid and AD pathology, with modest diagnostic accuracy (AUC = 0.601). NfL was increased in non-AD cases and in a subset of those with ADNC. Plasma biomarkers did not show changes in Lewy body disease (LBD), hippocampal sclerosis of aging (HS) or limbic-predominant age-related TDP-43 encephalopathy (LATE) unless ADNC was present. Higher levels of P-tau181, 231 and NfL predicted faster cognitive decline, as early as 10 years prior to autopsy, even among people with normal cognition or mild cognitive impairment. These results support plasma P-tau181 and 231 as diagnostic biomarkers related to ADNC that also can help to predict future cognitive decline, even in predementia stages. Although NfL was not consistently increased in plasma in AD and shows increases in several neurological disorders, it had utility to predict cognitive decline. Plasma Aβ42/40 as measured in this study was a relatively weak predictor of amyloid pathology, and different assay methods may be needed to improve on this. Additional plasma biomarkers are needed to detect the presence and impact of LBD and LATE pathology.