Reversible phosphorylation of Rpn1 regulates 26S proteasome assembly and function

Shu

et al. 2020

Preprint

Self Cite

Reversible phosphorylation has emerged as an important mechanism for regulating 26S proteasome function in health and disease. Over 100 phospho-tyrosine (pTyr) sites of the human proteasome have been detected, and yet their function and regulation remain poorly understood. Here we show that the 19S subunit Rpt2 is phosphorylated at Tyr439, a strictly conserved residue within the C-terminal HbYX motif of Rpt2 that is essential for 26S proteasome assembly. Unexpectedly, we found that Y439 phosphorylation depends on Rpt2 membrane localization mediated by its N-myristoylation. Multiple receptor tyrosine kinases (RTKs) can trigger Rpt2-Y439 phosphorylation by activating Src, a N-myristoylated tyrosine kinase. Src directly phosphorylates Rpt2-Y439 in vitro and negatively regulates 26S proteasome integrity and activity at cellular membranes, which can be reversed by the membrane-associated isoform of protein tyrosine phosphatase non-receptor type 2 (PTPN2). In H1975 lung cancer cells with activated Src, blocking Rpt2-Y439 phosphorylation by the Y439F mutation conferred partial resistance to the Src inhibitor saracatinib both in vitro and in a mouse xenograft tumor model, and caused significant changes of cellular responses to saracatinib at the proteome level. Our study has defined a novel mechanism involved in the spatial regulation of proteasome function and provided new insights into tyrosine kinase inhibitor-based anti-cancer therapies.

Section: Discussionsupporting

confidence: 75%

Section: Plasmids Shrnas and Sgrnasmentioning

confidence: 99%

Proteasome Regulation by Reversible Tyrosine Phosphorylation at the Membrane

Shu

et al. 2020

Preprint

Self Cite

Subcellular Biochemistry

“…Catalyzed by proteasome-associated phosphatases or kinases, phosphorylation is a more extensively studied post-translational modification in the proteasome, and affects many proteasome subunits (Iwafune et al 2002;Lu et al 2008;Kikuchi et al 2010;VerPlank and Goldberg 2017;Liu et al 2020;VerPlank et al 2019). The proteasome disassembles into the free CP and RP after treatment with alkaline phosphatase (Satoh et al 2001).…”

Section: Post-translational Modifications Of Proteasomementioning

confidence: 99%

Structure, Dynamics and Function of the 26S Proteasome

Mao

2020

The 26S proteasome is the most complex ATP-dependent protease machinery, of ~2.5 MDa mass, ubiquitously found in all eukaryotes. It selectively degrades ubiquitin-conjugated proteins and plays fundamentally indispensable roles in regulating almost all major aspects of cellular activities. To serve as the sole terminal “processor” for myriad ubiquitylation pathways, the proteasome evolved exceptional adaptability in dynamically organizing a large network of proteins, including ubiquitin receptors, shuttle factors, deubiquitinases, AAA-ATPase unfoldases, and ubiquitin ligases, to enable substrate selectivity and processing efficiency and to achieve regulation precision of a vast diversity of substrates. The inner working of the 26S proteasome is among the most sophisticated, enigmatic mechanisms of enzyme machinery in eukaryotic cells. Recent breakthroughs in three-dimensional atomic-level visualization of the 26S proteasome dynamics during polyubiquitylated substrate degradation elucidated an extensively detailed picture of its functional mechanisms, owing to progressive methodological advances associated with cryogenic electron microscopy (cryo-EM). Multiple sites of ubiquitin binding in the proteasome revealed a canonical mode of ubiquitin-dependent substrate engagement. The proteasome conformation in the act of substrate deubiquitylation provided insights into how the deubiquitylating activity of RPN11 is enhanced in the holoenzyme and is coupled to substrate translocation. Intriguingly, three principal modes of coordinated ATP hydrolysis in the heterohexameric AAA-ATPase motor were discovered to regulate intermediate functional steps of the proteasome, including ubiquitin-substrate engagement, deubiquitylation, initiation of substrate translocation and processive substrate degradation. The atomic dissection of the innermost working of the 26S proteasome opens up a new era in our understanding of the ubiquitin-proteasome system and has far-reaching implications in health and disease.

“…Importantly, inhibition can be fully reversed by supplementation of aspartate or pyruvate thus demonstrating adaptive metabolic fine-tuning of 26S proteasome function, which may have therapeutic implications. elegantly shown for the 19S subunits Rpt3 [51,52] and Rpn1 [53]. In addition, assembly chaperones regulate the formation of 26S proteasome complexes from the 20S catalytic core and 19S regulatory particles thus allowing timely adjustment of 26S proteasome activity to cellular demands [33,50].…”

Section: Discussionmentioning

confidence: 99%

Adaptive mitochondrial regulation of the proteasome

Meul

2020

Preprint

The proteasome is the main proteolytic system for targeted protein degradation in the cell. Its function is fine-tuned according to cellular needs. Regulation of proteasome function by mitochondrial metabolism, however, is unknown.Here, we demonstrate that mitochondrial dysfunction reduces the assembly and activity of the 26S proteasome in the absence of oxidative stress. Impaired respiratory complex I function leads to metabolic reprogramming of the Krebs cycle and deficiency in aspartate. Aspartate supplementation activates assembly and activity of 26S proteasomes via transcriptional activation of the proteasome assembly factors p28 and Rpn6. This metabolic adaptation of 26S proteasome function involves sensing of aspartate via the mTORC1 pathway. Metformin treatment of primary human cells similarly reduced assembly and activity of 26S proteasome complexes, which was fully reversible and rescued by supplementation of aspartate or pyruvate. Of note, respiratory dysfunction conferred resistance towards the proteasome inhibitor Bortezomib.Our study uncovers a fundamental novel mechanism of how mitochondrial metabolism adaptively adjusts protein degradation by the proteasome. It thus unravels unexpected consequences of defective mitochondrial metabolism in disease or drug-targeted mitochondrial reprogramming for proteasomal protein degradation in the cell. As metabolic inhibition of proteasome function can be alleviated by treatment with aspartate or pyruvate, our results also have therapeutic implications. 4 Keywords 26S proteasome, mitochondria, respiratory complex I, aspartate, metabolic reprogramming, proteasome assembly factors, pyruvate 5