Determining the architectures of macromolecular assemblies

To understand the workings of a living cell, we need to know the architectures of its macromolecular assemblies. Here we show how proteomic data can be used to determine such structures. The process involves the collection of sufficient and diverse high-quality data, translation of these data into spatial restraints, and an optimization that uses the restraints to generate an ensemble of structures consistent with the data. Analysis of the ensemble produces a detailed architectural map of the assembly. We developed our approach on a challenging model system, the nuclear pore complex (NPC). The NPC acts as a dynamic barrier, controlling access to and from the nucleus, and in yeast is a 50 MDa assembly of 456 proteins. The resulting structure, presented in an accompanying paper, reveals the configuration of the proteins in the NPC, providing insights into its evolution and architectural principles. The present approach should be applicable to many other macromolecular assemblies.

The molecular architecture of the nuclear pore complex

Nuclear pore complexes (NPCs) are proteinaceous assemblies of approximately 50 MDa that selectively transport cargoes across the nuclear envelope. To determine the molecular architecture of the yeast NPC, we collected a diverse set of biophysical and proteomic data, and developed a method for using these data to localize the NPC’s 456 constituent proteins (see the accompanying paper). Our structure reveals that half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. This scaffold forms an interlaced network that coats the entire curved surface of the nuclear envelope membrane within which the NPC is embedded. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold. The NPC consists of only a few structural modules that resemble each other in terms of the configuration of their homologous constituents, the most striking of these being a 16-fold repetition of ‘columns’. These findings provide clues to the evolutionary origins of the NPC.

Lysines in the tetramerization domain of p53 selectively modulate G1 arrest

<p>Functional in a tetrameric state, the protein product of the <i>p53</i> tumor suppressor gene confers its tumor-suppressive activity by transactivating genes which promote cell-cycle arrest, senescence, or programmed cell death. How p53 distinguishes between these divergent outcomes is still a matter of considerable interest. Here we discuss the impact of 2 mutations in the tetramerization domain that confer unique properties onto p53. By changing lysines 351 and 357 to arginine, thereby blocking all post-translational modifications of these residues, DNA binding and transcriptional regulation by p53 remain virtually unchanged. On the other hand, by changing these lysines to glutamine (2KQ-p53), thereby neutralizing their positive charge and potentially mimicking acetylation, p53 is impaired in the induction of cell cycle arrest and yet can still effectively induce cell death. Surprisingly, when 2KQ-p53 is expressed at high levels in H1299 cells, it can bind to and transactivate numerous p53 target genes including <i>p21</i>, but not others such as <i>miR-34a</i> and <i>cyclin G1</i> to the same extent as wild-type p53. Our findings show that strong induction of p21 is not sufficient to block H1299 cells in G1, and imply that modification of one or both of the lysines within the tetramerization domain may serve as a mechanism to shunt p53 from inducing cell cycle arrest.</p

Distinct Expression Profiles of p63 Variants during Urothelial Development and Bladder Cancer Progression

The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform–specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors