Programmed Ribosomal Frameshifting Mediates Expression of the α-Carboxysome.

Many bacteria employ a protein organelle, the carboxysome, to catalyze carbon dioxide fixation in the Calvin Cycle. Only 10 genes from Halothiobacillus neapolitanus are sufficient for heterologous expression of carboxysomes in Escherichia coli, opening the door to detailed mechanistic analysis of the assembly process of this complex (more than 200MDa). One of these genes, csoS2, has been implicated in assembly but ascribing a molecular function is confounded by the observation that the single csoS2 gene yields expression of two gene products and both display an apparent molecular weight incongruent with the predicted amino acid sequence. Here, we elucidate the co-translational mechanism responsible for the expression of the two protein isoforms. Specifically, csoS2 was found to possess -1 frameshifting elements that lead to the production of the full-length protein, CsoS2B, and a truncated protein, CsoS2A, which possesses a C-terminus translated from the alternate frame. The frameshifting elements comprise both a ribosomal slippery sequence and a 3' secondary structure, and ablation of either sequence is sufficient to eliminate the slip. Using these mutants, we investigated the individual roles of CsoS2B and CsoS2A on carboxysome formation. In this in vivo formation assay, cells expressing only the CsoS2B isoform were capable of producing intact carboxysomes, while those with only CsoS2A were not. Thus, we have answered a long-standing question about the nature of CsoS2 in this model microcompartment and demonstrate that CsoS2B is functionally distinct from CsoS2A in the assembly of α-carboxysomes.

Toxic fibrillar oligomers of amyloid-ß have cross-ß structure.

Although amyloid fibers are found in neurodegenerative diseases, evidence points to soluble oligomers of amyloid-forming proteins as the cytotoxic species. Here, we establish that our preparation of toxic amyloid-ß(1-42) (Abeta42) fibrillar oligomers (TABFOs) shares with mature amyloid fibrils the cross-ß structure, in which adjacent ß-sheets adhere by interpenetration of protein side chains. We study the structure and properties of TABFOs by powder X-ray diffraction, EM, circular dichroism, FTIR spectroscopy, chromatography, conformational antibodies, and celluar toxicity. In TABFOs, Abeta42 molecules stack into short protofilaments consisting of pairs of helical ß-sheets that wrap around each other to form a superhelix. Wrapping results in a hole along the superhelix axis, providing insight into how Abeta may form pathogenic amyloid pores. Our model is consistent with numerous properties of Abeta42 fibrillar oligomers, including heterogenous size, ability to seed new populations of fibrillar oligomers, and fiber-like morphology.

Atomic view of a toxic amyloid small oligomer.

Amyloid diseases, including Alzheimer's, Parkinson's, and the prion conditions, are each associated with a particular protein in fibrillar form. These amyloid fibrils were long suspected to be the disease agents, but evidence suggests that smaller, often transient and polymorphic oligomers are the toxic entities. Here, we identify a segment of the amyloid-forming protein αB crystallin, which forms an oligomeric complex exhibiting properties of other amyloid oligomers: ß-sheet-rich structure, cytotoxicity, and recognition by an oligomer-specific antibody. The x-ray-derived atomic structure of the oligomer reveals a cylindrical barrel, formed from six antiparallel protein strands, that we term a cylindrin. The cylindrin structure is compatible with a sequence segment from the ß-amyloid protein of Alzheimer's disease. Cylindrins offer models for the hitherto elusive structures of amyloid oligomers.

Modularity of a carbon-fixing protein organelle.

Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO(2)-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO(2). Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO(2) in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures.

Ribonuclease A suggests how proteins self-chaperone against amyloid fiber formation.

Genomic analyses have identified segments with high fiber-forming propensity in many proteins not known to form amyloid. Proteins are often protected from entering the amyloid state by molecular chaperones that permit them to fold in isolation from identical molecules; but, how do proteins self-chaperone their folding in the absence of chaperones? Here, we explore this question with the stable protein ribonuclease A (RNase A). We previously identified fiber-forming segments of amyloid-related proteins and demonstrated that insertion of these segments into the C-terminal hinge loop of nonfiber-forming RNase A can convert RNase A into the amyloid state through three-dimensional domain-swapping, where the inserted fiber-forming segments interact to create a steric zipper spine. In this study, we convert RNase A into amyloid-like fibers by increasing the loop length and hence conformational freedom of an endogenous fiber-forming segment, SSTSAASS, in the N-terminal hinge loop. This is accomplished by sandwiching SSTSAASS between inserted Gly residues. With these inserts, SSTSAASS is now able to form the steric zipper spine, allowing RNase A to form amyloid-like fibers. We show that these fibers contain RNase A molecules retaining their enzymatic activity and therefore native-like structure. Thus, RNase A appears to prevent fiber formation by limiting the conformational freedom of this fiber-forming segment from entering a steric zipper. Our observations suggest that proteins have evolved to self-chaperone by using similar protective mechanisms.

Identifying the amylome, proteins capable of forming amyloid-like fibrils.

The amylome is the universe of proteins that are capable of forming amyloid-like fibrils. Here we investigate the factors that enable a protein to belong to the amylome. A major factor is the presence in the protein of a segment that can form a tightly complementary interface with an identical segment, which permits the formation of a steric zipper-two self-complementary beta sheets that form the spine of an amyloid fibril. Another factor is sufficient conformational freedom of the self-complementary segment to interact with other molecules. Using RNase A as a model system, we validate our fibrillogenic predictions by the 3D profile method based on the crystal structure of NNQQNY and demonstrate that a specific residue order is required for fiber formation. Our genome-wide analysis revealed that self-complementary segments are found in almost all proteins, yet not all proteins form amyloids. The implication is that chaperoning effects have evolved to constrain self-complementary segments from interaction with each other.

Short protein segments can drive a non-fibrillizing protein into the amyloid state.

Protein fibrils termed amyloid-like are associated with numerous degenerative diseases as well as some normal cellular functions. Specific short segments of amyloid-forming proteins have been shown to form fibrils themselves. However, it has not been shown in general that these segments are capable of driving a protein from its native structure into the amyloid state. We applied the 3D profile method to identify fibril-forming segments within the amyloid-forming human proteins tau, alpha-synuclein, PrP prion and amyloid-beta. Ten segments, six to eight residues in length, were chosen and inserted into the C-terminal hinge loop of the highly constrained enzyme RNase A, and tested for fibril growth and Congo red birefringence. We find that all 10 unique inserts cause RNase A to form amyloid-like fibrils which display characteristic yellow to apple-green Congo red birefringence when observed with cross polarizers. These six to eight residue inserts can fibrillize RNase A and are sufficient for amyloid fibril spine formation.

A novel CYR61-triggered 'CYR61-alphavbeta3 integrin loop' regulates breast cancer cell survival and chemosensitivity through activation of ERK1/ERK2 MAPK signaling pathway.

The angiogenic inducer CYR61 is differentially overexpressed in breast cancer cells exhibiting high levels of Heregulin (HRG), a growth factor closely associated with a metastatic breast cancer phenotype. Here, we examined whether CYR61, independently of HRG, actively regulates breast cancer cell survival and chemosensitivity, and the pathways involved. Forced expression of CYR61 in HRG-negative MCF-7 cells notably upregulated the expression of its own integrin receptor alphavbeta3 (>200 times). Small peptidomimetic alphavbeta3 integrin antagonists dramatically decreased cell viability of CYR61-overexpressing MCF-7 cells, whereas control MCF-7/V remained insensitive. Mechanistically, functional blockade of alphavbeta3 specifically abolished CYR6-induced hyperactivation of ERK1/ERK2 MAPK, whereas the activation status of AKT did not decrease. Moreover, CYR61 overexpression rendered MCF-7 cells significantly resistant (>10-fold) to Taxol-induced cytotoxicity. Remarkably, alphavbeta3 inhibition converted the CYR61-induced Taxol-resistant phenotype into a hypersensitive one. Thus, the augmentation of Taxol-induced apoptotic cell death in the presence of alphavbeta3 antagonists demonstrated a strong synergism as verified by the terminal transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometric analysis for DNA content. Indeed, functional blockade of alphavbeta3, similarly to the pharmacological MAPK inhibitor U0126, synergistically increased both the proportion of CYR61-overexpressing breast cancer cells in the G2 phase of the cell cycle and the appearance of sub-G1 hypodiploid (apoptotic) cells caused by Taxol. Strikingly, CYR61 overexpression impaired the accumulation of wild-type p53 following Taxol exposure, while inhibition of alphavbeta3 or ERK1/ERK2 MAPK signalings completely restored Taxol-induced upregulation of p53. Moreover, antisense downregulation of CYR61 expression abolished the anchorage-independent growth of breast cancer cells engineered to overexpress HRG, and significantly increased their sensitivity to Taxol. Our data provide evidence that CYR61 is sufficient to promote breast cancer cell proliferation, cell survival, and Taxol resistance through a alphavbeta3-activated ERK1/ERK2 MAPK signaling. The identification of a 'CYR61-alphavbeta3 autocrine loop' in the epithelial compartment of breast carcinoma strongly suggests that targeting alphavbeta3 may simultaneously prevent breast cancer angiogenesis, growth, and chemoresistance.

Pharmacological inhibition of fatty acid synthase (FAS): a novel therapeutic approach for breast cancer chemoprevention through its ability to suppress Her-2/neu (erbB-2) oncogene-induced malignant transformation.

We designed our experiments to evaluate whether fatty acid synthase (FAS), a lipogenic enzyme linked to tumor virulence in population studies of human cancer, is necessary for the malignant transformation induced by Her-2/neu (erbB-2) oncogene, which is overexpressed not only in invasive breast cancer but also in premalignant atypical duct proliferations and in ductal carcinoma in situ of the breast. To avoid the genetic complexities associated with established breast cancer cell lines, we employed NIH-3T3 mouse fibroblasts engineered to overexpress human Her-2/neu coding sequence. NIH-3T3/Her-2 cells demonstrated a significant upregulation of FAS protein expression, which was dependent on the upstream activation of mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/AKT pathways. Remarkably, pharmacological FAS blockade using the mycotoxin cerulenin or the novel small compound C75 completely suppressed the state of Her-2/neu-induced malignant transformation by inhibiting the ability of NIH-3T3/Her-2 cells to grow under either anchorage-independent (i.e., to form colonies in soft agar) or low-serum monolayer conditions. Moreover, NIH-3T3/Her-2 fibroblasts were up to three times more sensitive to chemical FAS inhibitors relative to untransformed controls as determined by MTT-based cell viability assays. In addition, pharmacological FAS blockade preferentially induced apoptotic cell death of NIH-3T3/Her-2 fibroblasts, as determined by an ELISA for histone-associated DNA fragments and by the terminal deoxynucleotidyltransferase (TdT)-mediated nick end labeling assay (TUNEL). Interestingly, the degree of Her-2/neu oncogene expression in a panel of breast cancer cell lines was predictive of sensitivity to chemical FAS inhibitors-induced cytotoxicity, while low-FAS expressing and chemical FAS inhibitors-resistant MDA-MB-231 breast cancer cells became hypersensitive to FAS blockade when they were engineered to overexpress Her-2/neu. Our observations strongly suggest that inhibition of FAS activity may provide a new molecular avenue for chemotherapeutic prevention and/or treatment of Her-2/neu-related breast carcinomas.