Identification of a regulatory element for yeast tryptophan permease Tat2 ubiquitination using high hydrostatic pressure

ABSTRACT

Tryptophan uptake in the yeast Saccharomyces cerevisiae is extremely sensitive to high pressure; therefore, the growth of tryptophan auxotrophic strains is impaired. Degradation of tryptophan permease Tat2 is enhanced at 25?MPa, depending on Rsp5 ubiquitin ligase. Any defect in Tat2 ubiquitination confers high pressure growth capacity, which is a luminous phenotype of the yeast used to explore the mechanism by which Rsp5 mediates Tat2 ubiquitination. Here we show that the N-terminal four (K17, K20, K29, and K31) among five lysines are required for efficient Tat2 degradation under high pressure. We found that a domain spanning D70 to S76 is also critical for Tat2 degradation at 25?MPa probably because of the recognition by Bul1, an adaptor protein of Rsp5. Defects in Tat2 ubiquitination do not produce any remarkable mutant phenotype at 0.1?MPa. Therefore, we suggest that high pressure is a unique and advanced tool to explore ubiquitination-dependent Tat2 regulation.     

Synthetic approaches to constructing proteolysis targeting chimeras (PROTACs)

The development of various heterobifunctional constructs dubbed PRoteolysis-TArgeting Chimeras (PROTACs) has gained a significant impetus in the last few years. A viable alternative to the traditional occupancy-based inhibition of aberrantly hyperactive proteins, PROTACs operate by an event-based catalytic mechanism bringing together the protein of interest (POI, to be degraded) and E3 ubiquitin ligases. The formation of the ternary complex ‘POI–PROTAC–E3 ubiquitin ligase’ is the critical step which leads to the ubiquitination of the POI and its proteasomal degradation. The current Focused Review aims to highlight the syntheses of selected innovative PROTAC-type degraders of the therapeutically important protein targets as well as some notable chemical aspects of PROTAC construction. The overview is focusing on PROTACs aimed at recruiting Cereblon, the most exploited E3 ligase for targeted protein degradation.     

Ubiquitylation of Fe65 adaptor protein by neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) via the WW domain interaction with Fe65

Fe65 has been characterized as an adaptor protein, originally identified as an expressed sequence tag (EST) corresponding to an mRNA expressed at high levels in the rat brain. It contains one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1/PID2). As the neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) has a putative WW domain binding motif (⁷²PPLP⁷⁵) in the N-terminal domain, we hypothesized that Fe65 associates with Nedd4-2 through a WW domain interaction, which has the characteristics of E3 ubiquitin-protein ligase. In this paper, we present evidence for the interaction between Fe65 WW domain and Nedd4-2 through its specific motif, using a pull down approach and co-immunoprecipitation. Additionally, the co-localization of Fe65 and Nedd4-2 were observed via confocal microscopy. Co-localization of Fe65 and Nedd4-2 was disrupted by either the mutation of Fe65 WW domain or its putative binding motif of Nedd4-2. When the ubiquitin assay was performed, the interaction of Nedd4-2 (wt) with Fe65 is required for the cell apoptosis and the ubiquitylation of Fe65. We also observed that the ubiquitylation of Fe65 (wt) was augmented depending on Nedd4-2 expression levels, whereas the Fe65 WW domain mutant (W243KP245K) or the Nedd4-2 AL mutant (⁷²PPLP⁷⁵ was changed to ⁷²APLA⁷⁵) was under-ubiquitinated significantly. Thus, our observations implicated that the protein-protein interaction between the WW domain of Fe65 and the putative binding motif of Nedd4-2 down-regulates Fe65 protein stability and subcellular localization through its ubiquitylation, to contribute cell apoptosis.     

双光束光解离在牛泛素蛋白离子的自顶向下质谱分析中的应用研究

自顶向下(Top-down)质谱分析方法是将完整蛋白质离子碎片化,从而在分子水平上提供更加精准、丰富的与蛋白质结构相关的生物学信息.该文首次将3μm红外激光与210 nm紫外激光共同引入到傅里叶变换离子回旋共振质谱仪(FT-ICR MS)的分析池中,获得了牛泛素蛋白离子的自顶向下质谱.通过优化两束激光被引入的时间序列,...     

Brain site-specific proteome changes in aging-related dementia

This study is aimed at gaining insights into the brain site-specific proteomic senescence signature while comparing physiologically aged brains with aging-related dementia brains (for example, Alzheimer''s disease (AD)). Our study of proteomic differences within the hippocampus (Hp), parietal cortex (pCx) and cerebellum (Cb) could provide conceptual insights into the molecular mechanisms involved in aging-related neurodegeneration. Using an isobaric tag for relative and absolute quantitation (iTRAQ)-based two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS) brain site-specific proteomic strategy, we identified 950 proteins in the Hp, pCx and Cb of AD brains. Of these proteins, 31 were significantly altered. Most of the differentially regulated proteins are involved in molecular transport, nervous system development, synaptic plasticity and apoptosis. Particularly, proteins such as Gelsolin (GSN), Tenascin-R (TNR) and AHNAK could potentially act as novel biomarkers of aging-related neurodegeneration. Importantly, our Ingenuity Pathway Analysis (IPA)-based network analysis further revealed ubiquitin C (UBC) as a pivotal protein to interact with diverse AD-associated pathophysiological molecular factors and suggests the reduced ubiquitin proteasome degradation system (UPS) as one of the causative factors of AD.