Proteasome regulation,plant growth and stress tolerance

Plant cells contain a mixture of 26S and 20S proteasomes that mediate ubiquitin-dependent and ubiquitin-independent proteolysis, respectively. The 26S proteasome contains the 20S proteasome and one or two regulatory particles that are required for ubiquitin-dependent degradation. Comparative analyses of Arabidopsis proteasome mutants revealed that a decrease in 26S proteasome biogenesis causes heat shock hypersensitivity and reduced cell division rates that are compensated by increased cell expansion. Loss of 26S proteasome function also leads to an increased 20S proteasome biogenesis, which in turn enhances the cellular capacity to degrade oxidized proteins and thus increases oxidative stress tolerance. These findings suggest the intriguing possibility that 26S and 20S proteasome activities are regulated to control plant development and stress responses. This mini-review highlights some of the recent studies on proteasome regulation in plants.Key words: proteasome, cell division, ubiquitin-dependent proteolysis, ubiquitin-independent proteolysis, stress responses     

A screenable in vivo assay to study proteostasis networks in Caenorhabditis elegans

In eukaryotic cells, the ubiquitin/proteasome system (UPS) is a key determinant of proteostasis as it regulates the turnover of damaged proteins. However, it is still unclear how the UPS integrates intrinsic and environmental challenges to promote organismal development and survival. Here, we set up an in vivo degradation assay to facilitate the genetic identification of ubiquitin-dependent proteolysis pathways in the multicellular organism Caenorhabditis elegans. Using this assay, we found that mild induction of protein-folding stress, which is nontoxic for wild-type worms, strongly reduces ubiquitin-dependent protein turnover. Ubiquitin-mediated degradation is also reduced by metabolic stress, which correlates with life-span extension. Unlike other stress conditions, however, acute heat stress results in enhanced rather than reduced proteolysis. Intriguingly, our study provides the first evidence for the existence of tissue-specific degradation requirements because loss of key regulators of the UPS, such as proteasomal subunits, causes accumulation of the model substrate, depending on the tissue type. Thus, here we establish a screenable degradation assay that allows diverse genetic screening approaches for the identification of novel cell-type-specific proteostasis networks important for developmental processes, stress response, and aging, thereby substantially extending the work on recently described mechanistic UPS reporter studies.     

Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis

Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death （PCD）. However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 r_esistant11-1 （/br11-1）, which fails to generate reactive oxygen intermediates （ROIs）, is incapable of initiating PCD when the mutant is challenged by fumonisin B l （FB0, a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base 1 （LCB 1） subunit of serine palmitoyltransferase （SPT）, which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FBl. By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine- 1-phosphate in a dosedependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.     

High-level production of tetraacetyl phytosphingosine (TAPS) by combined genetic engineering of sphingoid base biosynthesis and L-serine availability in the non-conventional yeast Pichia ciferrii

The non-conventional yeast Pichia ciferrii is known to secrete the sphingoid long-chain base phytosphingosine in a tetraacetylated form (TAPS). Sphingolipids are important ingredients in cosmetic applications as they play important roles in human skin. Our work aimed to improve TAPS production by genetic engineering of P. ciferrii. In the first step we improved precursor availability by blocking degradation of L-serine, which is condensed with palmitoyl-CoA by serine palmitoyltransferase in the first committed step of sphingolipid biosynthesis. Successive deletion of two genes, SHM1 and SHM2, encoding L-serine hydroxymethyltransferases, and of CHA1 encoding L-serine deaminase, resulted in a strain producing 65 mg((TAPS))g(-1)((cdw)), which is a threefold increase in comparison with the parental strain. Attempts to increase the metabolic flux into and through the L-serine biosynthesis pathway did not improve TAPS production. However, genetic engineering of the sphingolipid pathway further increased secretion of TAPS. Blocking of sphingoid long-chain base phosphorylation by deletion of the LCB kinase gene PcLCB4 resulted in a further increase in TAPS production by 78% and significant secretion of the direct precursor of phytosphingosine, sphinganin, in a triacetylated form (TriASa). Overproduction of two serine palmitoyltransferase subunits, Lcb1 and Lcb2, together with a deletion of the gene ORM12 encoding a putative negative regulator of sphingolipid synthesis resulted in a strain producing 178 mg((TAPS))g(-1)((cdw)). Additional overproduction of the C4-hydroxylase Syr2 converting sphinganine to phytosphingosine reduced TriASa production and further improved TAPS production. The final recombinant P. ciferrii strain produced up to 199 mg((TAPS))g(-1)((cdw)) with a maximal production rate of 8.42 mg×OD(600nm)(-1)h(-1) and a titer of about 2 g L(-1), and should be applicable for industrial TAPS production.     

PROTAC及其在肿瘤治疗方面的应用

肿瘤的发生与肿瘤特异癌蛋白的表达密切相关,调控这些蛋白质的降解已成为肿瘤治疗的一种新方法。泛素介导的蛋白质降解通路控制着真核细胞内绝大多数蛋白质的选择性降解。近年来,一种人工合成的蛋白靶向降解嵌合分子(proteolysis targeting chimeric molecule,PROTAC),能通过利用细胞固有的泛素-蛋白酶体系统调控靶向蛋白质降解。本文总结了PROTAC的相关研究进展,虽然对PROTAC技术的研究还有很多尚待解决的问题,但其作为肿瘤治疗的新手段具有很大的潜力。     

Intracellular localization of the proteasome in response to stress conditions

The ubiquitin–proteasome system fulfills an essential role in regulating protein homeostasis by spatially and temporally controlling proteolysis in an ATP- and ubiquitin-dependent manner. However, the localization of proteasomes is highly variable under diverse cellular conditions. In yeast, newly synthesized proteasomes are primarily localized to the nucleus during cell proliferation. Yeast proteasomes are transported into the nucleus through the nuclear pore either as immature subcomplexes or as mature enzymes via adapter proteins Sts1 and Blm10, while in mammalian cells, postmitotic uptake of proteasomes into the nucleus is mediated by AKIRIN2, an adapter protein essentially required for nuclear protein degradation. Stressful growth conditions and the reversible halt of proliferation, that is quiescence, are associated with a decline in ATP and the reorganization of proteasome localization. Cellular stress leads to proteasome accumulation in membraneless granules either in the nucleus or in the cytoplasm. In quiescence, yeast proteasomes are sequestered in an ubiquitin-dependent manner into motile and reversible proteasome storage granules in the cytoplasm. In cancer cells, upon amino acid deprivation, heat shock, osmotic stress, oxidative stress, or the inhibition of either proteasome activity or nuclear export, reversible proteasome foci containing polyubiquitinated substrates are formed by liquid–liquid phase separation in the nucleus. In this review, we summarize recent literature revealing new links between nuclear transport, ubiquitin signaling, and the intracellular organization of proteasomes during cellular stress conditions.     

E3 ubiquitin ligases and mitosis: embracing the complexity

Faithful division of eukaryotic cells requires temporal and spatial coordination of morphological transitions, which ensures that the newly replicated copies of the genome are equally distributed into the two daughter cells during mitosis. One of the mechanisms ensuring the fidelity of mitotic progression is targeted, ubiquitin-dependent proteolysis of key regulators. E3-ubiquitin ligase complexes are crucial components in this pathway because they specifically select the relevant ubiquitination substrates. Cullin-based E3-ligases, such as Cul3, have recently emerged as crucial regulators of mitosis.     

Down-regulation of model yeast proteins by ubiquitin-dependent proteolysis

Ubiquitination is a versatile tool used by all eukaryotic organisms for controlling the stability, function, and intracellular localization of a multitude of proteins. I will attempt to bring together our recent data on the down-regulation of two yeast model proteins, the galactose transporter Gal2 and fructose-1,6-bisphosphatase, by ubiquitin-dependent proteolysis triggered by the addition of easily fermentable carbon sources.     

Role for de novo sphingoid base biosynthesis in the heat-induced transient cell cycle arrest of Saccharomyces cerevisiae

The recent findings of sphingolipids as potential mediators of yeast heat stress responses led us to investigate their possible role in the heat-induced cell cycle arrest and subsequent recovery. The sphingolipid-deficient yeast strain 7R4 was found to lack the cell cycle arrest seen in the isogenic wild type. Furthermore, strain lcb1-100, which harbors a temperature-sensitive serine palmitoyltransferase, lacked increased de novo generated sphingoid bases upon heat stress. Importantly, this strain was found to lack the transient heat-induced G0/G1 arrest. These results indicate a role for sphingolipids and specifically those generated in the de novo pathway in the cell cycle arrest response to heat. To determine the bioactive sphingolipid regulating this response, an analysis of key mutants in the sphingolipid biosynthetic and degradation pathways was performed. Strains deleted in sphingoid base kinases, sphingoid phosphate phosphatase, lyase, or dihydrosphingosine hydroxylase were found to display the cell cycle arrest. Also, the knockout of a fatty acyl elongation enzyme, which severely attenuates ceramide production, displayed the arrest. These experiments suggested that the active species for cell cycle arrest were the sphingoid bases. In further support of these findings, exogenous phytosphingosine (10 microM) was found to induce transient arrest. Stearylamine did not induce an arrest, demonstrating chemical specificity, and L-erythro- was not as potent as D-erythro-dihydrosphingosine showing stereospecificity. To investigate a possible arrest mechanism, we studied the hyperstable Cln3 (Cln3-1) strain LDW6A that has been previously shown to be resistant to heat stress-induced cell cycle arrest. The strain containing Cln3-1 was found to be resistant to cell cycle arrest induced by exogenous phytosphingosine, indicating that Cln3 acts downstream of the sphingoid bases in this response. Interestingly, cell cycle recovery from the transient arrest was found to be dependent upon the sphingoid base kinases (LCB4, LCB5). Overall, this combination of genetic and pharmacologic results demonstrates a role for de novo sphingoid base biosynthesis by serine palmitoyltransferase in the transient G0/G1 arrest mediated through Cln3 via a novel mechanism.     

In vivo function of the proteasome in the ubiquitin pathway.

A major eukaryotic proteolytic system is known to require the covalent attachment of ubiquitin to substrates prior to their degradation, yet the proteinase involved remains poorly defined. The proteasome, a large conserved multi-subunit protein complex of the cytosol and the nucleus, has been implicated in a variety of cellular functions. It is shown here that a yeast mutant with a defective proteasome fails to degrade proteins which are subject to ubiquitin-dependent proteolysis in wild-type cells. Thus, the proteasome is part of the ubiquitin system and mediates the degradation of ubiquitin-protein conjugates in vivo.     

Uncoupling ubiquitin-protein conjugation from ubiquitin-dependent proteolysis by use of beta, gamma-nonhydrolyzable ATP analogues.

Pathways of ubiquitin-dependent protein degradation have in common two requirements for ATP. Ubiquitin activation by the enzyme E1 is accompanied by ATP hydrolysis to yield AMP and PPi, and during conjugate breakdown, the ubiquitin-dependent protease hydrolyzes ATP to ADP and Pi. We show here that either of two beta, gamma-nonhydrolyzable ATP analogues, 5'-adenylyl imidodiphosphate or 5'-adenylyl methylenediphosphate, can support ubiquitin-protein conjugation. With the ubiquitin-dependent protease, however, neither analogue could substitute for ATP. Thus, the substitution of a beta, gamma-nonhydrolyzable analogue for ATP offers a simple method to uncouple ubiquitin conjugation from proteolysis in crude systems. On the basis of pyrophosphate exchange kinetics, E1 has apparent Km and Vmax values that are similar for ATP and the analogues, but substrate inhibition by 5'-adenylyl methylenediphosphate made use of the beta, gamma-imido analogue preferable. In one application, beta, gamma-imido-ATP was used in combination with ubiquitin aldehyde (an inhibitor of ubiquitin-protein isopeptidases) to establish that several unfolded RNase A derivatives are recognized equally as ubiquitination substrates. This result extends an earlier study [Dunten, R. L., & Cohen, R. E. (1989) J. Biol. Chem. 264, 16739-16747] to show that conjugate yields, upon which relative ubiquitination rates were based, were not influenced by differential ubiquitin-dependent proteolysis. In a second application, ATP and beta, gamma-imido-ATP were compared in a pulse-chase experiment to investigate the contributions of ATP-dependent proteolysis and isopeptidase activities to conjugate stability.     

The separation and direct detection of ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and evaporative light-scattering detection

Sphingolipids are an important class of lipids due to their role as biologically active molecules and as intracellular second messengers. Sphingolipid metabolites are involved in a wide variety of important biological processes including signal transduction and growth regulation. Simple, quantitative analytical methods are needed to assay these complex lipids, in order to study their biological functions. The current methods used to quantify ceramides and long-chain sphingoid bases are primarily based on derivatization with uv or fluorescent tags and with radioactive-based enzymatic assays. A method was developed to separate ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and detect them directly with evaporative light-scattering detection. Ceramides and the sphingoid bases phytosphingosine, dihydrosphingosine, sphingosine, and sphingosine 1-phosphate were resolved with a rapid and quantitative assay in the nanomole range. Yeast extracts grown to various time points were assayed for ceramide and sphingoid bases using a simple, isocratic HPLC system. Both ceramide and phytosphingosine, the primary sphingoid base present in yeast cell extracts, were detected in yeast cell extracts. Phytosphingosine was resolved as a sharp peak with the addition of triethylamine and formic acid modifiers to a chloroform/ethanol mobile phase. This method demonstrates the first direct assay of both ceramides and sphingoid bases.     

ATP-stimulated degradation of endogenous proteins in cell-free extracts of BHK 21/C13 fibroblasts. A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins.

Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogenous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogenous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rates of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.     

Sphingolipid transport in eukaryotic cells

Sphingolipids constitute a sizeable fraction of the membrane lipids in all eukaryotes and are indispensable for eukaryotic life. First of all, the involvement of sphingolipids in organizing the lateral domain structure of membranes appears essential for processes like protein sorting and membrane signaling. In addition, recognition events between complex glycosphingolipids and glycoproteins are thought to be required for tissue differentiation in higher eukaryotes and for other specific cell interactions. Finally, upon certain stimuli like stress or receptor activation, sphingolipids give rise to a variety of second messengers with effects on cellular homeostasis. All sphingolipid actions are governed by their local concentration. The intricate control of their intracellular topology by the proteins responsible for their synthesis, hydrolysis and intracellular transport is the topic of this review.     

Hemin inhibits ubiquitin-dependent proteolysis in both a higher plant and yeast

In eukaryotes, a major route for ATP-dependent protein breakdown proceeds through covalent intermediates of target proteins destined for degradation and the highly conserved, 76 amino acid protein ubiquitin. In rabbit reticulocytes, it has been shown that hemin effectively inhibits this pathway by blocking the catabolism of ubiquitin-protein conjugates [KI = 25 microM (Haas, A. L., & Rose, I. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6845-6848)]. Here, we demonstrate that hemin is also an effective inhibitor of the ubiquitin-dependent proteolytic pathway in both a higher plant, oats (Avena sativa), and yeast (Saccharomyces cerevisiae). Hemin inhibits all stages of the pathway in vitro, including ATP-dependent formation of ubiquitin-protein conjugates, disassembly of conjugates by ubiquitin-protein lyase(s) (or isopeptidases), and degradation of ubiquitin-protein conjugates by ATP-dependent protease(s). Using ubiquitin-125I-lysozyme conjugates synthesized in vitro as substrates, we determined the specific effects of hemin on the rates of disassembly and degradation separately. The concentration of hemin required for half-maximal inhibition of both processes was identical in each species, approximately 60 microM in oats and approximately 50 microM in yeast. Similar inhibitory effects were observed when two hemin analogues, mesoheme or protoporphyrin IX, were employed. These results demonstrate that the effect of hemin on ubiquitin-dependent proteolysis is not restricted to erythroid cells and as a result hemin may be a useful tool in studies of this pathway in all eukaryotic cells. These results also question models where hemin serves as a specific negative modulator of proteolysis in erythroid cells.