The proteasome antechamber maintains substrates in an unfolded state

Eukaryotes and archaea use a protease called the proteasome that has an integral role in maintaining cellular function through the selective degradation of proteins. Proteolysis occurs in a barrel-shaped 20S core particle, which in Thermoplasma acidophilum is built from four stacked homoheptameric rings of subunits, α and β, arranged α(7)β(7)β(7)α(7) (ref. 5). These rings form three interconnected cavities, including a pair of antechambers (formed by α(7)β(7)) through which substrates are passed before degradation and a catalytic chamber (β(7)β(7)) where the peptide-bond hydrolysis reaction occurs. Although it is clear that substrates must be unfolded to enter through narrow, gated passageways (13?? in diameter) located on the α-rings, the structural and dynamical properties of substrates inside the proteasome antechamber remain unclear. Confinement in the antechamber might be expected to promote folding and thus impede proteolysis. Here we investigate the folding, stability and dynamics of three small protein substrates in the antechamber by methyl transverse-relaxation-optimized NMR spectroscopy. We show that these substrates interact actively with the antechamber walls and have drastically altered kinetic and equilibrium properties that maintain them in unstructured states so as to be accessible for hydrolysis.     

A cryptic protease couples deubiquitination and degradation by the proteasome

The 26S proteasome is responsible for most intracellular proteolysis in eukaryotes. Efficient substrate recognition relies on conjugation of substrates with multiple ubiquitin molecules and recognition of the polyubiquitin moiety by the 19S regulatory complex--a multisubunit assembly that is bound to either end of the cylindrical 20S proteasome core. Only unfolded proteins can pass through narrow axial channels into the central proteolytic chamber of the 20S core, so the attached polyubiquitin chain must be released to allow full translocation of the substrate polypeptide. Whereas unfolding is rate-limiting for the degradation of some substrates and appears to involve chaperone-like activities associated with the proteasome, the importance and mechanism of degradation-associated deubiquitination has remained unclear. Here we report that the POH1 (also known as Rpn11 in yeast) subunit of the 19S complex is responsible for substrate deubiquitination during proteasomal degradation. The inability to remove ubiquitin can be rate-limiting for degradation in vitro and is lethal to yeast. Unlike all other known deubiquitinating enzymes (DUBs) that are cysteine proteases, POH1 appears to be a Zn(2+)-dependent protease.     

A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes

The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. It comprises one catalytic 20S proteasome and two axially positioned 19S regulatory complexes. The 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, alpha1-7beta1-7beta1-7alpha1-7 (refs 4, 5), but the mechanism responsible for the assembly of such a complex structure remains elusive. Here we report two chaperones, designated proteasome assembling chaperone-1 (PAC1) and PAC2, that are involved in the maturation of mammalian 20S proteasomes. PAC1 and PAC2 associate as heterodimers with proteasome precursors and are degraded after formation of the 20S proteasome is completed. Overexpression of PAC1 or PAC2 accelerates the formation of precursor proteasomes, whereas knockdown by short interfering RNA impairs it, resulting in poor maturation of 20S proteasomes. Furthermore, the PAC complex provides a scaffold for alpha-ring formation and keeps the alpha-rings competent for the subsequent formation of half-proteasomes. Thus, our results identify a mechanism for the correct assembly of 20S proteasomes.     

Quantitative dynamics and binding studies of the 20S proteasome by NMR

The machinery used by the cell to perform essential biological processes is made up of large molecular assemblies. One such complex, the proteasome, is the central molecular machine for removal of damaged and misfolded proteins from the cell. Here we show that for the 670-kilodalton 20S proteasome core particle it is possible to overcome the molecular weight limitations that have traditionally hampered quantitative nuclear magnetic resonance (NMR) spectroscopy studies of such large systems. This is achieved by using an isotope labelling scheme where isoleucine, leucine and valine methyls are protonated in an otherwise highly deuterated background in concert with experiments that preserve the lifetimes of the resulting NMR signals. The methodology has been applied to the 20S core particle to reveal functionally important motions and interactions by recording spectra on complexes with molecular weights of up to a megadalton. Our results establish that NMR spectroscopy can provide detailed insight into supra-molecular structures over an order of magnitude larger than those routinely studied using methodology that is generally applicable.     

ClpS is an essential component of the N-end rule pathway in Escherichia coli

The N-end rule states that the half-life of a protein is determined by the nature of its amino-terminal residue. Eukaryotes and prokaryotes use N-terminal destabilizing residues as a signal to target proteins for degradation by the N-end rule pathway. In eukaryotes an E3 ligase, N-recognin, recognizes N-end rule substrates and mediates their ubiquitination and degradation by the proteasome. In Escherichia coli, N-end rule substrates are degraded by the AAA + chaperone ClpA in complex with the ClpP peptidase (ClpAP). Little is known of the molecular mechanism by which N-end rule substrates are initially selected for proteolysis. Here we report that the ClpAP-specific adaptor, ClpS, is essential for degradation of N-end rule substrates by ClpAP in bacteria. ClpS binds directly to N-terminal destabilizing residues through its substrate-binding site distal to the ClpS-ClpA interface, and targets these substrates to ClpAP for degradation. Degradation by the N-end rule pathway is more complex than anticipated and several other features are involved, including a net positive charge near the N terminus and an unstructured region between the N-terminal signal and the folded protein substrate. Through interaction with this signal, ClpS converts the ClpAP machine into a protease with exquisitely defined specificity, ideally suited to regulatory proteolysis.     

A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal

The 26S proteasome is the chief site of regulatory protein turnover in eukaryotic cells. It comprises one 20S catalytic complex (composed of four stacked rings of seven members) and two axially positioned 19S regulatory complexes (each containing about 18 subunits) that control substrate access to the catalytic chamber. In most cases, targeting to the 26S proteasome depends on tagging of the substrate with a specific type of polyubiquitin chain. Recognition of this signal is followed by substrate unfolding and translocation, which are presumably catalysed by one or more of six distinct AAA ATPases located in the base-a ring-like 19S subdomain that abuts the axial pore of the 20S complex and exhibits chaperone activity in vitro. Despite the importance of polyubiquitin chain recognition in proteasome function, the site of this signal's interaction with the 19S complex has not been identified previously. Here we use crosslinking to a reactive polyubiquitin chain to show that a specific ATPase subunit, S6' (also known as Rpt5), contacts the bound chain. The interaction of this signal with 26S proteasomes is modulated by ATP hydrolysis. Our results suggest that productive recognition of the proteolytic signal, as well as proteasome assembly and substrate unfolding, are ATP-dependent events.     

Effect of non-contacted bases on the affinity of 434 operator for 434 repressor and Cro

The repressor of phage 434 binds to six operator sites on the phage chromosome. A comparison of the sequences of these 14-base-pair (bp) operator sites reveals a striking pattern: at five of the six sites, the symmetrically arrayed outer eight base pairs (four in each half-site) are identical and the remaining site differs at only one position (Fig. 1b). In contrast, the sequences of the inner four base pairs are highly variable. Crystallographic analysis of the repressor-operator complex shows that at each half-site, the 'recognition alpha-helix' of the repressor is positioned in the major groove such that it could contact the outermost five base pairs, but not the innermost two (Fig. 1a). We show in this paper that the sequence of the central base pairs of the operator (two in each half-site) have a significant role in determining operator affinity for repressor, despite the evidence presented here and in the accompanying paper that these base pairs are not contacted by repressor. We also show that these central base pairs influence operator affinity for Cro, a second gene regulatory protein encoded by phage 434. We discuss the likely structural basis for this evidently indirect, but sequence-dependent, effect of the central base pairs of the operator on its affinity for the two regulatory proteins.     

Activator-assisted electroless deposition of copper nanostructured films

This paper showed simple and effective synthesis of copper nanoparticles within controlled diameter using direct electroless deposition on glass substrates, following the sensitization and activation steps. Electroless-deposited metals, such as Cu, Co, Ni, and Ag, and their alloys had many advantages in micro- and nanotechnologies. The structural, morphological, and optical properties of copper deposits were characterized using X-ray diffraction (XRD), atomic force microscopy (AFM), and UV-Vis spectroscopy. The structural data was further analyzed using the Rietveld refinement program. Structural studies reveal that the deposited copper prefers a (111) orientation. AFM studies suggest the deposited materials form compact, uniform, and nanocrystalline phases with a high tendency to self-organize. The data show that the particle size can be controlled by controlling the activator concentration. The absorption spectra of the as-deposited copper nanoparticles reveal that the plasmonic peak broadens and exhibits a blue shift with decreasing particle size.     

26S蛋白酶体调控微管蛋白的降解

为了探究微管蛋白在植物细胞中的降解机制,本文利用微管解聚药物、蛋白酶体抑制剂MG132结合遗传学和分子生物学等体内体外研究技术发现微管蛋白TUA和TUB是通过泛素化/26S蛋白酶体途径降解的,同时在26S蛋白酶体调节亚基RPN10突变体rpn10-1的植株中,TUA的降解与野生型相比没有显著差别,但是TUB的降解在突变体中被严重削弱,使得TUB在突变体中过量积累.此外,在突变体中分别过量表达TUA和TUB,对转基因植株鉴定表明过量表达TUA促进突变体根的生长而过量表达TUB严重抑制了突变体的生长,说明在突变体中TUA/TUB的比例是失衡的,过量的TUB蛋白对植物的生长是不利的.以上结果表明,26S蛋白酶体可以调控微管蛋白TUA和TUB的降解,蛋白酶体调控亚基RPN10可以通过调控TUB的稳定性来影响植物的生长和发育.     

Targets of the cyclin-dependent kinase Cdk1

The events of cell reproduction are governed by oscillations in the activities of cyclin-dependent kinases (Cdks). Cdks control the cell cycle by catalysing the transfer of phosphate from ATP to specific protein substrates. Despite their importance in cell-cycle control, few Cdk substrates have been identified. Here, we screened a budding yeast proteomic library for proteins that are directly phosphorylated by Cdk1 in whole-cell extracts. We identified about 200 Cdk1 substrates, several of which are phosphorylated in vivo in a Cdk1-dependent manner. The identities of these substrates reveal that Cdk1 employs a global regulatory strategy involving phosphorylation of other regulatory molecules as well as phosphorylation of the molecular machines that drive cell-cycle events. Detailed analysis of these substrates is likely to yield important insights into cell-cycle regulation.     

Novel flow behaviors induced by a solid particle in nanochannels: Poiseuille and Couette

We conducted a molecular dynamics(MD)simulation to address the novel flow behaviors induced by a solid particle in nanochannels.Two basic flows,i.e.,Poiseuille and Couette,are involved in this study.For Poiseuille flow,the distribution of number density exhibits fluctuations in the center of channel and near the walls,which are caused by the strong interactions from the atoms of particle and walls.For stronger external driving forces,the fluid atoms move toward the center of channel and some cavities appear in the fluidic zone.Greater external driving forces and bigger particles make the fluid moving quickly in nanochannels.For Couette flow,the particle rotates under the velocity difference among particle atoms in the shear flows.The fluid atoms near the walls become infrequent and move to the center of channel.The velocities of the centrally-located fluid atoms decrease owing to the particle,resulting in an untypical non-linear Couette flow.In summary,the solid particle brings new fluid–solid interface and interactions,which induce several novel behaviors in nanochannel flows.     

阶乘矩识别不同规律控制过程

探讨阶乘矩识别不同规律控制下过程的可行性,以期为DNA编码识别等提供可能的工具。讨论了阶乘矩描述序列过程精度与事件数的关系,在此基础上考察了等几率随机过程、高斯分布随机过程、确定性过程的阶乘矩和以一种过程为背景混合另一种过程作为扰动的混合过程的阶乘矩。发现加入确定性过程将在背景基础上引起明显的阶乘矩行为的变化。因此阶乘矩可以有效地把确定性过程从随机控制过程背景或另外一种确定性过程背景中有效地识别出来。大量的非线性统计研究结果表明,DNA分子中编码区中碱基没有长程关联,服从随机分布,而非编码区碱基序列有长程关联,具有自相似结构特征,服从完全不同于随机的分布规律,因此阶乘矩应该是很好的识别编码区的备选工具。     

Proteasome subunit Rpn13 is a novel ubiquitin receptor

Proteasomal receptors that recognize ubiquitin chains attached to substrates are key mediators of selective protein degradation in eukaryotes. Here we report the identification of a new ubiquitin receptor, Rpn13/ARM1, a known component of the proteasome. Rpn13 binds ubiquitin through a conserved amino-terminal region termed the pleckstrin-like receptor for ubiquitin (Pru) domain, which binds K48-linked diubiquitin with an affinity of approximately 90 nM. Like proteasomal ubiquitin receptor Rpn10/S5a, Rpn13 also binds ubiquitin-like (UBL) domains of UBL-ubiquitin-associated (UBA) proteins. In yeast, a synthetic phenotype results when specific mutations of the ubiquitin binding sites of Rpn10 and Rpn13 are combined, indicating functional linkage between these ubiquitin receptors. Because Rpn13 is also the proteasomal receptor for Uch37, a deubiquitinating enzyme, our findings suggest a coupling of chain recognition and disassembly at the proteasome.     

The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase

Many biological processes, such as development and cell cycle progression are tightly controlled by selective ubiquitin-dependent degradation of key substrates. In this pathway, the E3-ligase recognizes the substrate and targets it for degradation by the 26S proteasome. The SCF (Skp1-Cul1-F-box) and ECS (Elongin C-Cul2-SOCS box) complexes are two well-defined cullin-based E3-ligases. The cullin subunits serve a scaffolding function and interact through their C terminus with the RING-finger-containing protein Hrt1/Roc1/Rbx1, and through their N terminus with Skp1 or Elongin C, respectively. In Caenorhabditis elegans, the ubiquitin-ligase activity of the CUL-3 complex is required for degradation of the microtubule-severing protein MEI-1/katanin at the meiosis-to-mitosis transition. However, the molecular composition of this cullin-based E3-ligase is not known. Here we identified the BTB-containing protein MEL-26 as a component required for degradation of MEI-1 in vivo. Importantly, MEL-26 specifically interacts with CUL-3 and MEI-1 in vivo and in vitro, and displays properties of a substrate-specific adaptor. Our results suggest that BTB-containing proteins may generally function as substrate-specific adaptors in Cul3-based E3-ubiquitin ligases.     

泌阳凹陷毕店地区核桃园组核三段沉积特征与含油性研究

毕店地区位于多物源交汇带,沉积相带变化快,各个物源作用范围界定不清,受不同相带控制的砂体发育规律认识不清。通过对研究区沉积特征及含油性研究,对确定砂体规模和分布范围、寻找岩性-构造复合型油气藏有重要现实意义。本文在岩心、录井、测井及测试分析等资料研究基础上,认为研究区西部、北部、东北主要发育曲流河三角洲沉积体系,南部发育扇三角洲沉积体系以及东南部发育重力流与湖泊沉积体系;研究层段的基准面经历过6次上升和3次下降过程,在基准面相对较低时期,其主体沉积微相为水下分流河道、河口坝(前缘砂),沉积砂体较厚,水下分流河道延伸范围广,河道分支较多;在基准面相对较高时期,其主体沉积微相为远砂坝(席状砂)、湖相,沉积砂体薄,水下分流河道规模小、数量少,远砂坝及席状砂大面积发育。结合构造、生产动态数据,认为研究区油藏属于构造油气藏、岩性油气藏和复合油气藏,油气聚集受构造情况、优势沉积相,基准面升降等控制明显。     

Materials for Lithium Batteries Prepared via Mechanochemical Route

1 Results Nanostructured materials are currently of interest for lithium-ion batteries due to relevant demands for high-rate performance batteries and the aspect of structural stability (reversibility) under charge-discharge processes.Decreasing of particle size facilitates the reducing of diffuse paths for lithium ions as compared with micron-sized materials and the increasing of surface contact between electrode and electrolyte leading to acceleration of ionic transport and of charge-discharge process...