High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris.

Solution NMR approaches for establishing specificity of weak heterodimerization of membrane proteins.

Solution NMR provides a powerful approach for detecting complex formation involving weak to moderate intermolecular affinity. However, solution NMR has only rarely been used to detect complex formation between two membrane proteins in model membranes. The impact of specific binding on the NMR spectrum of a membrane protein can be difficult to distinguish from spectral changes that are induced by nonspecific binding and/or by changes that arise from forced cohabitation of the two proteins in a single model membrane assembly.

Dependency of γ-secretase complex activity on the structural integrity of the bilayer.

γ-secretase is a membrane protein complex associated with the production of Aβ peptides that are pathogenic in Alzheimer's disease. We have characterized the activity of γ-secretase complexes under a variety of detergent solubilization and reconstitution conditions, and the structural state of proteoliposomes by electron microscopy. We found that γ-secretase activity is highly dependent on the physical state or integrity of the membrane bilayer--partial solubilization may increase activity while complete solubilization will abolish it.

Hybrid molecular structure of the giant protease tripeptidyl peptidase II.

Tripeptidyl peptidase II (TPP II) is the largest known eukaryotic protease (6 MDa). It is believed to act downstream of the 26S proteasome, cleaving tripeptides from the N termini of longer peptides, and it is implicated in numerous cellular processes. Here we report the structure of Drosophila TPP II determined by a hybrid approach.

Structural studies of the transmembrane C-terminal domain of the amyloid precursor protein (APP): does APP function as a cholesterol sensor?

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein.

Regulation of gamma-secretase activity in Alzheimer's disease.

The gamma-secretase complex is an intramembrane aspartyl protease that cleaves its substrates along their transmembrane regions. Sequential proteolytic processing of amyloid precursor protein by beta- and gamma-secretase produces amyloid beta-peptides, which are the major components of amyloid plaques in the brains of Alzheimer's disease patients. The gamma-secretase complex is therefore believed to be critical in the pathogenesis of Alzheimer's disease.

Proteasome assembly triggers a switch required for active-site maturation.

The processing of propeptides and the maturation of 20S proteasomes require the association of beta rings from two half proteasomes. We propose an assembly-dependent activation model in which interactions between helix (H3 and H4) residues of the opposing half proteasomes are prerequisite for appropriate positioning of the S2-S3 loop; such positioning enables correct coordination of the active-site residue needed for propeptide cleavage. Mutations of H3 or H4 residues that participate in the association of two half proteasomes inhibit activation and prevent, in nearly all cases, the formation of full proteasomes.

The discovery and role of CD147 as a subunit of gamma-secretase complex.

Gamma-secretase is a membrane protein complex with unusual aspartyl protease activity that cleaves a variety of type I transmembrane proteins, such as APP, Notch and E-cadherin, within their transmembranous regions. Gamma-secretase was first recognized because of its role in the production of Abeta peptides that are pathogenic in Alzheimer's disease. There is overwhelming evidence demonstrating that four components, presenilin, nicastrin, APH-1 and PEN-2, are necessary and sufficient for gamma-secretase activity.

CD147 is a regulatory subunit of the gamma-secretase complex in Alzheimer's disease amyloid beta-peptide production.

gamma-Secretase is a membrane protein complex that cleaves the beta-amyloid precursor protein (APP) within the transmembrane region, after prior processing by beta-secretase, producing amyloid beta-peptides Abeta(40) and Abeta(42). Errant production of Abeta-peptides that substantially increases Abeta(42) production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1, which contains the proteolytic active site, and three other membrane proteins [nicastrin, anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 (PEN-2)] are required to form the core of the active gamma-secretase complex.

Structural genomics of membrane proteins.

Improvements in the fields of membrane-protein molecular biology and biochemistry, technical advances in structural data collection and processing, and the availability of numerous sequenced genomes have paved the way for membrane-protein structural genomics efforts. There has been significant recent progress, but various issues essential for high-throughput membrane-protein structure determination remain to be resolved.

Crystal structures of the Rhodococcus proteasome with and without its pro-peptides: implications for the role of the pro-peptide in proteasome assembly.

To understand the role of the pro-peptide in proteasome assembly, we have determined structures of the Rhodococcus proteasome and a mutant form that prevents the autocatalytic removal of its pro-peptides. The structures reveal that the pro-peptide acts as an assembly-promoting factor by linking its own beta-subunit to two adjacent alpha-subunits, thereby providing a molecular explanation for the observed kinetics of proteasome assembly. The Rhodococcus proteasome has been found to have a substantially smaller contact region between alpha-subunits compared to those regions in the proteasomes of Thermoplasma, yeast, and mammalian cells, suggesting that a smaller contact area between alpha-subunits is likely the structural basis for the Rhodococcus alpha-subunits not assembling into alpha-rings when expressed alone.