bims-proarb Biomed News
on Proteostasis in aging and regenerative biology
Issue of 2021‒10‒31
fifteen papers selected by
Rich Giadone
Harvard University
  1. Protein clearance strategies for disease intervention.
  2. Pharmacological targeting of endoplasmic reticulum stress in disease.
  3. Macroautophagy and normal aging of the nervous system: Lessons from animal models.
  4. A single-cell transcriptomic atlas of primate pancreatic islet aging.
  5. Prolonged disturbance of proteostasis induces cellular senescence via temporal mitochondrial dysfunction and subsequent mitochondrial accumulation in human fibroblasts.
  6. Autophagy coordinates chondrocyte development and early joint formation in zebrafish.
  7. Temporal proteomics during neurogenesis reveals large-scale proteome and organelle remodeling via selective autophagy.
  8. Alzheimer's drugs: Does reducing amyloid work?
  9. The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag.
  10. Induction of UPR Promotes Interferon Response to Inhibit PRRSV Replication via PKR and NF-κB Pathway.
  11. Protocol to alter a protein's phase separation capacity to control cell fate transitions.
  12. Resveratrol prevents haloperidol-induced mitochondria dysfunction through the induction of autophagy in SH-SY5Y cells.
  13. Deficient Chaperone-Mediated Autophagy Promotes Inflammation and Atherosclerosis.
  14. Lactoferrin promotes the autophagy activity during osteoblast formation via BCL2-Beclin1 signaling.
  15. Inhibition of ubiquitin-specific protease 13-mediated degradation of Raf1 kinase by Spautin-1 has opposing effects in naïve and primed pluripotent stem cells.
  1. J Neural Transm (Vienna). 2021 Oct 23.
    Protein clearance strategies for disease intervention.
    Franziska Hommen, Saygın Bilican, David Vilchez.
      Protein homeostasis, or proteostasis, is essential for cell function and viability. Unwanted, damaged, misfolded and aggregated proteins are degraded by the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. Growing evidence indicates that alterations in these major proteolytic mechanisms lead to a demise in proteostasis, contributing to the onset and development of distinct diseases. Indeed, dysregulation of the UPS or autophagy is linked to several neurodegenerative, infectious and inflammatory disorders as well as cancer. Thus, modulation of protein clearance pathways is a promising approach for therapeutics. In this review, we discuss recent findings and open questions on how targeting proteolytic mechanisms could be applied for disease intervention.
    Keywords:  Aging; Alzheimer’s disease; Amyotrophic lateral sclerosis; Autophagy; Cancer; Huntington’s disease; Parkinson’s disease; Proteasome; Proteostasis
    DOI:  https://doi.org/10.1007/s00702-021-02431-y
  2. Nat Rev Drug Discov. 2021 Oct 26.
    Pharmacological targeting of endoplasmic reticulum stress in disease.
    Stefan J Marciniak, Joseph E Chambers, David Ron.
      The accumulation of misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress, resulting in activation of the unfolded protein response (UPR) that aims to restore protein homeostasis. However, the UPR also plays an important pathological role in many diseases, including metabolic disorders, cancer and neurological disorders. Over the last decade, significant effort has been invested in targeting signalling proteins involved in the UPR and an array of drug-like molecules is now available. However, these molecules have limitations, the understanding of which is crucial for their development into therapies. Here, we critically review the existing ER stress and UPR-directed drug-like molecules, highlighting both their value and their limitations.
    DOI:  https://doi.org/10.1038/s41573-021-00320-3
  3. Cell Stress. 2021 Oct;5(10): 146-166
    Macroautophagy and normal aging of the nervous system: Lessons from animal models.
    Emmanouela Kallergi, Vassiliki Nikoletopoulou.
      Aging represents a cumulative form of cellular stress, which is thought to challenge many aspects of proteostasis. The non-dividing, long-lived neurons are particularly vulnerable to stress, and, not surprisingly, even normal aging is highly associated with a decline in brain function in humans, as well as in other animals. Macroautophagy is a fundamental arm of the proteostasis network, safeguarding proper protein turnover during different cellular states and against diverse cellular stressors. An intricate interplay between macroautophagy and aging is beginning to unravel, with the emergence of new tools, including those for monitoring autophagy in cultured neurons and in the nervous system of different organisms in vivo. Here, we review recent findings on the impact of aging on neuronal integrity and on neuronal macroautophagy, as they emerge from studies in invertebrate and mammalian models.
    Keywords:  aging; macroautophagy; nervous system; proteostasis
    DOI:  https://doi.org/10.15698/cst2021.10.257
  4. Natl Sci Rev. 2021 Feb;8(2): nwaa127
    A single-cell transcriptomic atlas of primate pancreatic islet aging.
    Jingyi Li, Yuxuan Zheng, Pengze Yan, Moshi Song, Si Wang, Liang Sun, Zunpeng Liu, Shuai Ma, Juan Carlos Izpisua Belmonte, Piu Chan, Qi Zhou, Weiqi Zhang, Guang-Hui Liu, Fuchou Tang, Jing Qu.
      Aging-related degeneration of pancreatic islet cells contributes to impaired glucose tolerance and diabetes. Endocrine cells age heterogeneously, complicating the efforts to unravel the molecular drivers underlying endocrine aging. To overcome these obstacles, we undertook single-cell RNA sequencing of pancreatic islet cells obtained from young and aged non-diabetic cynomolgus monkeys. Despite sex differences and increased transcriptional variations, aged β-cells showed increased unfolded protein response (UPR) along with the accumulation of protein aggregates. We observed transcriptomic dysregulation of UPR components linked to canonical ATF6 and IRE1 signaling pathways, comprising adaptive UPR during pancreatic aging. Notably, we found aging-related β-cell-specific upregulation of HSP90B1, an endoplasmic reticulum-located chaperone, impeded high glucose-induced insulin secretion. Our work decodes aging-associated transcriptomic changes that underlie pancreatic islet functional decay at single-cell resolution and indicates that targeting UPR components may prevent loss of proteostasis, suggesting an avenue to delaying β-cell aging and preventing aging-related diabetes.
    Keywords:  aging; islet; primate; single-cell RNA sequencing; β-cell
    DOI:  https://doi.org/10.1093/nsr/nwaa127
  5. FEBS J. 2021 Oct 24.
    Prolonged disturbance of proteostasis induces cellular senescence via temporal mitochondrial dysfunction and subsequent mitochondrial accumulation in human fibroblasts.
    Yasuhiro Takenaka, Ikuo Inoue, Takanari Nakano, Masaaki Ikeda, Yoshihiko Kakinuma.
      Proteolytic activity declines with age, resulting in the accumulation of aggregated proteins in aged organisms. To investigate how disturbance in proteostasis causes cellular senescence, we developed a stress-induced premature senescence (SIPS) model, in which normal human fibroblast MRC-5 cells were treated with the proteasome inhibitor MG132 or the V-ATPase inhibitor bafilomycin A1 (BAFA1) for 5 days. Time-course studies revealed a significant increase in intracellular reactive oxygen species (ROS) and mitochondrial superoxide during and after drug treatment. Mitochondrial membrane potential initially decreased, suggesting temporal mitochondrial dysfunction during drug treatment, but was restored along with mitochondrial accumulation after drug treatment. AMP-activated protein kinase alpha (AMPKα) was notably activated during treatment; thereafter, intracellular ATP levels significantly increased. SIPS induction by MG132 or BAFA1 was partially attenuated by co-treatment with vitamin E or rapamycin, in which the levels of ROS, mitochondrial accumulation, and protein aggregates were suppressed, implying the critical involvement of oxidative stress and mitochondrial function in SIPS progression. Rapamycin co-treatment also augmented the expression of HSP70 and activation of AKT, which could recover proteostasis and promote cell survival, respectively. Our study proposes a possible pathway from the disturbed proteostasis to cellular senescence via excess ROS production as well as functional and quantitative changes in mitochondria.
    Keywords:  DNA damage response; aggregate; lysosome; oxidative stress; proteasome
    DOI:  https://doi.org/10.1111/febs.16249
  6. FASEB J. 2021 Nov;35(11): e22002
    Autophagy coordinates chondrocyte development and early joint formation in zebrafish.
    Joanna J Moss, Martina Wirth, Sharon A Tooze, Jon D Lane, Chrissy L Hammond.
      Autophagy is a catabolic process responsible for the removal of waste and damaged cellular components by lysosomal degradation. It plays a key role in fundamental cell processes, including ER stress mitigation, control of cell metabolism, and cell differentiation and proliferation, all of which are essential for cartilage cell (chondrocyte) development and survival, and for the formation of cartilage. Correspondingly, autophagy dysregulation has been implicated in several skeletal disorders such as osteoarthritis and osteoporosis. To test the requirement for autophagy during skeletal development in zebrafish, we generated an atg13 CRISPR knockout zebrafish line. This line showed a complete loss of atg13 expression, and restricted autophagic activity in vivo. In the absence of autophagy, chondrocyte maturation was accelerated, with chondrocytes exhibiting signs of premature hypertrophy. Focussing on the jaw element, autophagy disruption affected joint articulation causing restricted mouth opening. This gross behavioural phenotype corresponded with a failure to thrive, and death in homozygote atg13 nulls within 17 days. Taken together, our results are consistent with autophagy contributing to the timely regulation of chondrocyte maturation and for extracellular matrix formation.
    Keywords:  Atg13; autophagy; chondrocytes; joints; zebrafish
    DOI:  https://doi.org/10.1096/fj.202101167R
  7. Mol Cell. 2021 Oct 15. pii: S1097-2765(21)00800-5. [Epub ahead of print]
    Temporal proteomics during neurogenesis reveals large-scale proteome and organelle remodeling via selective autophagy.
    Alban Ordureau, Felix Kraus, Jiuchun Zhang, Heeseon An, Sookhee Park, Tim Ahfeldt, Joao A Paulo, J Wade Harper.
      Cell state changes are associated with proteome remodeling to serve newly emergent cell functions. Here, we show that NGN2-driven conversion of human embryonic stem cells to induced neurons (iNeurons) is associated with increased PINK1-independent mitophagic flux that is temporally correlated with metabolic reprogramming to support oxidative phosphorylation. Global multiplex proteomics during neurogenesis revealed large-scale remodeling of functional modules linked with pluripotency, mitochondrial metabolism, and proteostasis. Differentiation-dependent mitophagic flux required BNIP3L and its LC3-interacting region (LIR) motif, and BNIP3L also promoted mitophagy in dopaminergic neurons. Proteomic analysis of ATG12-/- iNeurons revealed accumulation of endoplasmic reticulum, Golgi, and mitochondria during differentiation, indicative of widespread organelle remodeling during neurogenesis. This work reveals broad organelle remodeling of membrane-bound organelles during NGN2-driven neurogenesis via autophagy, identifies BNIP3L's central role in programmed mitophagic flux, and provides a proteomic resource for elucidating how organelle remodeling and autophagy alter the proteome during changes in cell state.
    Keywords:  autophagy; iNeurons; mitophagy; proteomics
    DOI:  https://doi.org/10.1016/j.molcel.2021.10.001
  8. Science. 2021 Oct 29. 374(6567): 544-545
    Alzheimer's drugs: Does reducing amyloid work?
    Madhav Thambisetty, Robert Howard, M Maria Glymour, Lon S Schneider.
      
    DOI:  https://doi.org/10.1126/science.abl8366
  9. Nat Commun. 2021 Oct 26. 12(1): 6173
    The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag.
    Indrajit Sahu, Sachitanand M Mali, Prasad Sulkshane, Cong Xu, Andrey Rozenberg, Roni Morag, Manisha Priyadarsini Sahoo, Sumeet K Singh, Zhanyu Ding, Yifan Wang, Sharleen Day, Yao Cong, Oded Kleifeld, Ashraf Brik, Michael H Glickman.
      The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins.
    DOI:  https://doi.org/10.1038/s41467-021-26427-0
  10. Front Microbiol. 2021 ;12 757690
    Induction of UPR Promotes Interferon Response to Inhibit PRRSV Replication via PKR and NF-κB Pathway.
    Zhenbang Zhu, Panrao Liu, Lili Yuan, Zhengmin Lian, Danhe Hu, Xiaohui Yao, Xiangdong Li.
      Porcine reproductive and respiratory syndrome virus (PRRSV) was previously shown to induce a certain level of cellular stress during viral replication. Unfolded protein response (UPR) is a cellular stress response responsible for coping with stress and cellular survival. However, the pathway leading to the induction of UPR that may influence PRRSV replication is still unknown. Here, we found that PRRSV infection induced UPR prior to interferon response. Induction of UPR significantly enhanced the expression of interferon and interferon-related genes, thus leading to the suppression of PRRSV infection. Next, we explored the underlying mechanisms of UPR-induced antiviral response. We found that induction of UPR promoted the expression of protein kinase R (PKR), and PKR was highly correlated with the reduction of PRRSV replication. Furthermore, tunicamycin stimulation and PKR overexpression activated NF-κB and interferon response at the early stage of PRRSV infection, thus reinforcing the expression of type I interferons and proinflammatory cytokines and leading to inhibition of PRRSV. In addition, PRRSV nsp4 was shown to reduce the expression of PKR. These findings might have implications for our understandings of the host's immune mechanism against PRRSV and a new strategy of PRRSV to evade the host antiviral immunity.
    Keywords:  Nsp4; PKR; PRRSV; interferon response; unfolded protein response
    DOI:  https://doi.org/10.3389/fmicb.2021.757690
  11. STAR Protoc. 2021 Dec 17. 2(4): 100887
    Protocol to alter a protein's phase separation capacity to control cell fate transitions.
    Qian Ma, Jia Wang, Haopeng Yu, Junyi Sun, Jun Sun, Jiahao Chen, Yangyinhui Yu, Peihang Fang, Qi Tian, Junjun Ding.
      Phase separation of proteins regulates transcription. Here, we present a protocol to manipulate phase separation capacity of a protein. We use this protocol to disrupt phase separation by mutating residues at intrinsically disordered regions (IDRs). Further, we rescue the disabled phase separation by fusing an IDR known to drive phase separation. Phase separation promotes cell fate transitions, whereas disruption of phase attenuates the transitions. The major challenge is how to effectively predict mutation residues. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).
    Keywords:  Cell Biology; Cell-based Assays; Microscopy; Molecular Biology; Molecular/Chemical Probes; Protein expression and purification; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2021.100887
  12. Neurotoxicology. 2021 Oct 22. pii: S0161-813X(21)00127-3. [Epub ahead of print]87 231-242
    Resveratrol prevents haloperidol-induced mitochondria dysfunction through the induction of autophagy in SH-SY5Y cells.
    Minmin Hu, Ruiqi Wang, Xi Chen, Mingxuan Zheng, Peng Zheng, Zehra Boz, Renxian Tang, Kuiyang Zheng, Yinghua Yu, Xu-Feng Huang.
      BACKGROUND: Haloperidol is a commonly used antipsychotic drug and may increase neuronal oxidative stress associated with the side effects, including tardive dyskinesia and neurite withdraw. Autophagy plays a protective role in response to the accumulated reactive oxygen species (ROS) induced mitochondria damage. Resveratrol is an antioxidant compound having neuroprotective effects; however, it is unknown if resveratrol may stimulate autophagy and decrease mitochondria damage induced by haloperidol.HYPOTHESIS: We hypothesis that resveratrol stimulates the autophagic process and protects mitochondria lesion induced by haloperidol.
    METHODS: MitoSOX™ Red Mitochondrial Superoxide Indicator and MitoTracker™ Green FM staining were used to measure the amount of the mitochondria ROS production and mitochondria mass in human SH-SY5Y cells treated with haloperidol and/or resveratrol. Autophagic related dyes and Western blot were applied to study the autophagic process and related protein expression. Besides, tandem monomeric mRFP-GFP-LC3 was used to investigate the fusion of autophagosome and lysosome. Transmission electron microscopy was used to investigate the mitochondrial and autophagic ultrastructures with or without haloperidol and resveratrol treatment.
    RESULTS: Haloperidol administration significantly increased mitochondria ROS and mitochondrial mass, indicating the increase of mitochondria dysfunction. Although haloperidol increased the autophagosomes and lysosome formation, the autophagosome-lysosome fusion and degradation were impaired. This was because we found an increased p62 after haloperidol treatment, an indication of autophagy incompletion. Importantly, resveratrol promoted the degradation of p62, upregulated the formation of autophagolysosome, and reversed haloperidol-induced mitochondria damage.
    CONCLUSION: These results collectively suggest that resveratrol may be introduced as a protective compound against haloperidol-induced mitochondria impairment and aberrant autophagy.
    Keywords:  Autophagy; Haloperidol; Mitochondria ROS; Resveratrol
    DOI:  https://doi.org/10.1016/j.neuro.2021.10.007
  13. Circ Res. 2021 Oct 27.
    Deficient Chaperone-Mediated Autophagy Promotes Inflammation and Atherosclerosis.
    Qiao Lei, Jing Ma, Zihao Zhang, Wenhai Sui, Chungang Zhai, Dan Xu, Zunzhe Wang, Huixia Lu, Meng Zhang, Cheng Zhang, Wenqiang Chen, Yun Zhang.
      Rationale: The NLRP3 inflammasome is an important driver of atherosclerosis. Our previous study shows that chaperone-mediated autophagy (CMA), one of the main lysosomal degradative process, has a regulatory role in lipid metabolism of macrophage. However, whether the NLRP3 inflammasome is regulated by CMA and the role of CMA in atherosclerosis remain unclear. Objective: To determine the role of CMA in the regulation of NLRP3 inflammasome and atherosclerosis. Methods and Results: The expression of CMA marker, lysosome associated membrane protein type 2A (LAMP-2A), was first analyzed in ApoE-/- mouse aortas and human coronary atherosclerotic plaques and a significant down-regulation of LAMP-2A in advanced atherosclerosis in both mice and human was observed. To selectively block CMA, we generated macrophage-specific conditional LAMP-2A-knockout mouse strains in C57BL/6 mice and ApoE-/- mice. Deletion of macrophage LAMP-2A accelerated atherosclerotic lesion formation in the aortic root and the whole aorta in ApoE-/- mice. Mechanistically, LAMP-2A deficiency promoted NLRP3 inflammasome activation and subsequent release of mature IL-1β in macrophages and atherosclerotic plaques. Furthermore, gain-of-function studies verified that restoration of LAMP-2A levels in LAMP-2A-deficient macrophages greatly attenuated NLRP3 inflammasome activation. Importantly, we identified the NLRP3 protein as a CMA substrate and demonstrated that LAMP-2A deficiency did not affect the NLRP3 mRNA levels but hindered degradation of the NLRP3 protein through CMA pathway. Conclusions: CMA function becomes impaired during the progression of atherosclerosis, which increases NLRP3 inflammasome activation and secretion of IL-1β, promoting vascular inflammation and atherosclerosis progression. Our study unveils a new mechanism by which NLRP3 inflammasome is regulated in macrophages and atherosclerosis, thus providing a new insight into the role of autophagy-lysosomal pathway in atherosclerosis. Pharmacological activation of CMA may provide a novel therapeutic strategy for atherosclerosis and other NLRP3 inflammasome/IL-1β-driven diseases.
    DOI:  https://doi.org/10.1161/CIRCRESAHA.121.318908
  14. Mol Biol Rep. 2021 Oct 29.
    Lactoferrin promotes the autophagy activity during osteoblast formation via BCL2-Beclin1 signaling.
    Dianshan Ke, Xinwen Wang, Yinquan Lin, Shengwang Wei.
      BACKGROUND: Lactoferrin, as the main component of milk, can maintain osteoblast formation, which is conducive to the prevention and treatment of osteoporosis. Lactoferrin also serves as an autophagy regulator, especially in osteoblasts. This study aimed to explore the significance of autophagy in osteoblast formation regulated by lactoferrin and the internal mechanism.METHODS AND RESULTS: In this study, we firstly explored the roles of lactoferrin in the autophagy activity of primary osteoblasts (LC3 transformation rate, autophagosome formation). Subsequently, we further investigated the effects of lactoferrin on the BCL2 expression and BCL2-Beclin1 complex. Ultimately, the significance of BCL2 overexpression and Beclin1 silencing on lactoferrin-regulated osteoblast autophagy and osteogenic parameters (ALP activity and mRNA expression of PCNA, Col1, BGLAP and OPN) was observed by gene processing, respectively. Our results showed that lactoferrin enhanced the autophagy activity of osteoblasts. Importantly, lactoferrin inhibited BCL2 expression and the co-immunoprecipitation of BCL2 and Beclin1 in osteoblasts. Moreover, lactoferrin-promoted autophagy and osteogenic parameters was reversed by BCL2 overexpression or Beclin1 silencing in osteoblasts.
    CONCLUSIONS: In conclusion, lactoferrin can inhibit BCL2 expression in osteoblasts, further enhancing Beclin1-dependent autophagy activation.
    Keywords:  Autophagy; BCL2; Beclin1; Lactoferrin; Osteoblasts
    DOI:  https://doi.org/10.1007/s11033-021-06866-0
  15. J Biol Chem. 2021 Oct 21. pii: S0021-9258(21)01138-8. [Epub ahead of print] 101332
    Inhibition of ubiquitin-specific protease 13-mediated degradation of Raf1 kinase by Spautin-1 has opposing effects in naïve and primed pluripotent stem cells.
    Xin Wang, Xiaoxiao Wang, Xinbao Zhang, Yan Zhang, Zhenhua Zhu, Yuting Li, Meng Zhang, Junxiang Ji, Yang Yu, Shou-Dong Ye.
      Embryonic stem cells (ESCs) are progenitor cells that retain the ability to differentiate into various cell types and are necessary for tissue repair. Improving cell culture conditions to maintain the pluripotency of ESCs in vitro is an urgent problem in the field of regenerative medicine. Here, we reveal that Spautin-1, a specific small molecule inhibitor of ubiquitin-specific protease (USP) family members USP10 and USP13, promotes the maintenance of self-renewal and pluripotency of mouse ESCs in vitro. Functional studies reveal that only knockdown of USP13, but not USP10, is capable of mimicking the function of Spautin-1. Mechanistically, we demonstrate that USP13 physically interacts with, deubiquitinates, and stabilizes serine/threonine kinase Raf1 and thereby sustains Raf1 protein at the posttranslational level to activate the FGF/MEK/ERK pro-differentiation signaling pathway in naïve mouse ESCs. In contrast, in primed mouse epiblast stem cells and human induced-pluripotent stem cells, the addition of Spautin-1 had an inhibitory effect on Raf1 levels, but USP13 overexpression promoted self-renewal. The addition of a MEK inhibitor impaired the effect of USP13 upregulation in these cells. These findings provide new insights into the regulatory network of naïve and primed pluripotency as a result of Raf1 deubiquitylation.
    Keywords:  Embryonic stem cells; Raf1; Self-renewal; Spautin-1; USP13
    DOI:  https://doi.org/10.1016/j.jbc.2021.101332
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