Search

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

Organization into polarized three-dimensional structures defines whether epithelial cells are normal or malignant. In a model of morphogenesis, we show that inhibiting key signaling pathways in human breast cancer cells leads to "phenotypic reversion" of the malignant cells. Using architecture as an endpoint, we report that, in all cases, signaling through Raf/MEK/ERK disrupted tissue polarity via matrix metalloproteinase9 (MMP9) activity. Induction of Raf or activation of an engineered, functionally inducible MMP9 in nonmalignant cells led to loss of tissue polarity, and reinitiated proliferation. Conversely, inhibition of Raf or MMP9 with small molecule inhibitors or shRNAs restored the ability of cancer cells to form polarized quiescent structures. Silencing MMP9 expression also reduced tumor growth dramatically in a murine xenograft model. LC-MS/MS analysis comparing conditioned medium from nonmalignant cells with or without active MMP9 revealed laminin 111 (LM1) as an important target of MMP9. LM1 has been implicated in acinar morphogenesis; thus, its degradation by MMP9 provides a mechanism for loss of tissue polarity and reinitiation of growth associated with MMP9 activity. These findings underscore the importance of the dynamic reciprocity between the extracellular matrix integrity, tissue polarity, and Raf/MEK/ERK and MMP9 activities, providing an axis for either tissue homeostasis or malignant progression.

This review discusses the peroxisome biogenesis mediated by more than 20 peroxins involved in peroxisomal protein import and biogenesis. Results reveal that protein-protein interactions among peroxins are involved in the peroxisomal protein import and biogenesis of peroxisomal membrane proteins.

Pichia pastoris cells were studied by examining the kinetic and morphological intermediates in the vacuolar deterioration of peroxisomes through macroautophagy and microautophagy. The vacuolar membrane and peroxisomal matrix were then labeled with N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide and a fusion protein containing Ser-Lys-Leu and analyzed via fluorescence microscopy. The inhibitors influencing various steps of microautophagy did not impede the delivery of peroxisome to the vacuole.

In meiosis I, homologous chromosomes pair, recombine and segregate to opposite poles. These events and subsequent meiosis II ensure that each of the four meiotic products has one complete set of chromosomes. In this study, the meiotic pairing and segregation of a trisomic chromosome in a diploid (2n + 1) yeast strain was examined. We find that trivalent pairing and segregation is the favored arrangement. However, insertions near the centromere in one of the trisomic chromosomes leads to preferential pairing and segregation of the 'like' centromeres of the remaining two chromosomes, suggesting that bivalent-univalent pairing and segregation is favored for this region.

Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown. We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay. Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation. The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs. By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap. Coimmuno-precipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes. These data, taken together, suggest that Pex19p may have a chaperone-like role at the peroxisome membrane and that it is not the receptor for targeting of iPMPs to the peroxisome. Key words: organelle biogenesis * protein localization * peroxin * chaperone * mPTS receptor

Research was conducted to characterize a novel peroxin, Pex22p, from Pichia pastoris. The subcellular localization, targeting signal, interacting partners, functions and topology of the protein were analyzed. Results provide evidence of the roles of Pex4p and Pex22p in peroxisomal matrix protein import. Findings also demonstrate that the proteins are conserved in Saccharomyces cerevisiae where the hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.