Structural insights into the functional cycle of the ATPase module of the 26S proteasome.

In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA+ ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA+ ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.

Aggregation and chemical modification of monoclonal antibodies under upstream processing conditions.

PURPOSE: To investigate antibody stability and formation of modified species under upstream processing conditions. METHODS: The stability of 11 purified monoclonal human IgG1 and IgG4 antibodies, including an IgG1-based bispecific CrossMab, was compared in downscale mixing stress models. One of these molecules was further evaluated in realistic bioreactor stress models and in cell culture fermentations. Analytical techniques include size exclusion chromatography (SEC), turbidity measurements, cation exchange chromatography (cIEX), dynamic light scattering (DLS) and differential scanning calorimetry (DSC). RESULTS: Sensitivity in downscale stress models varies among antibodies and results in formation of high molecular weight (HMW) aggregates. Stability is increased in cell culture medium and in bioreactors. Media components stabilizing the proteins were identified. Extensive chemical modifications were detected both in stress models as well as during production of antibodies in cell culture fermentations. CONCLUSIONS: Protective compounds must be present in chemically defined fermentation media in order to stabilize antibodies against the formation of HMW aggregates. An increase in chemical modifications is detectable in bioreactor stress models and over the course of cell culture fermentations; this increase is dependent on the expression rate, pH, temperature and fermentation time. Consequently, product heterogeneity increases during upstream processing, and this compromises the product quality.

Recent advances in the structural biology of the 26S proteasome.

There is growing appreciation for the fundamental role of structural dynamics in the function of macromolecules. In particular, the 26S proteasome, responsible for selective protein degradation in an ATP dependent manner, exhibits dynamic conformational changes that enable substrate processing. Recent cryo-electron microscopy (cryo-EM) work has revealed the conformational dynamics of the 26S proteasome and established the function of the different conformational states. Technological advances such as direct electron detectors and image processing algorithms allowed resolving the structure of the proteasome at atomic resolution. Here we will review those studies and discuss their contribution to our understanding of proteasome function.

Generation of a highly active folding enzyme by combining a parvulin-type prolyl isomerase from SurA with an unrelated chaperone domain.

Parvulins are small prolyl isomerases and serve as catalytic domains of folding enzymes. SurA (survival protein A) from the periplasm of Escherichia coli consists of an inactive (Par1) and an active (Par2) parvulin domain as well as a chaperone domain. In the absence of the chaperone domain, the folding activity of Par2 is virtually abolished. We created a chimeric protein by inserting the chaperone domain of SlyD, an unrelated folding enzyme from the FKBP family, into a loop of the isolated Par2 domain of SurA. This increased its folding activity 450-fold to a value higher than the activity of SurA, in which Par2 is linked with its natural chaperone domain. In the presence of both the natural and the foreign chaperone domain, the folding activity of Par2 was 1500-fold increased. Related and unrelated chaperone domains thus are similarly efficient in enhancing the folding activity of the prolyl isomerase Par2. A sequence analysis of various chaperone domains suggests that clusters of exposed methionine residues in mobile chain regions might be important for a generic interaction with unfolded protein chains. This binding is highly dynamic to allow frequent transfer of folding protein chains between chaperone and catalytic domains.