bims-proarb 2022-09-04 papers

bims-proarb

Biomed News

on Proteostasis in aging and regenerative biology

Issue of 2022–09–04
nine papers selected by
Rich Giadone, Harvard University

Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors.
Oxidation of two cysteines within yeast Hsp70 impairs proteostasis while directly triggering an Hsf1-dependent cytoprotective response.
The Endoplasmic Reticulum and the Fidelity of Nascent Protein Localization.
LRP1 is a neuronal receptor for α-synuclein uptake and spread.
Editorial: Utility of protein aggregation assays from laboratory to clinical application.
The molecular and functional changes of Neural stem cells in Alzheimer's disease: Can they be reinvigorated to conduct Neurogenesis.
Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development.
Computationally-Aided Modeling of Hsp70-Client Interactions: Past, Present, and Future.
Pathological structural conversion of α-synuclein at the mitochondria induces neuronal toxicity.

J Biol Chem. 2022 Aug 25. pii: S0021-9258(22)00866-3. [Epub ahead of print] 102423

Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors.

Ya-Juan Wang, Xiao-Jing Di, Ting-Wei Mu.

  Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system. Maintenance of GABAA receptor protein homeostasis (proteostasis) in cells utilizing its interacting proteins is essential for the function of GABAA receptors. However, how the proteostasis network orchestrates GABAA receptor biogenesis in the endoplasmic reticulum (ER) is not well understood. Here, we employed a proteomics-based approach to systematically identify the interactomes of GABAA receptors. We carried out a quantitative immunoprecipitation-tandem mass spectrometry (IP-MS/MS) analysis utilizing stable isotope labeling by amino acids in cell culture (SILAC). Further, we performed comparative proteomics by using both wild type α1 subunit and a misfolding-prone α1 subunit carrying the A322D variant as the bait proteins. We identified 125 interactors for wild type α1-containing receptors, 105 proteins for α1(A322D)-containing receptors, and 54 overlapping proteins within these two interactomes. Our bioinformatics analysis identified potential GABAA receptor proteostasis network components, including chaperones, folding enzymes, trafficking factors, and degradation factors, and we assembled a model of their potential involvement in the cellular folding, degradation, and trafficking pathways for GABAA receptors. Additionally, we verified endogenous interactions between α1 subunits and selected interactors by using co-immunoprecipitation in mouse brain homogenates. Moreover, we showed that TRIM21, an E3 ubiquitin ligase, positively regulated the degradation of misfolding-prone α1(A322D) subunits selectively. This study paves the way for understanding the molecular mechanisms as well as fine-tuning of GABAA receptor proteostasis to ameliorate related neurological diseases such as epilepsy.

Keywords:  GABA(A) receptor; SILAC; assembly; degradation; epilepsy; folding; interactome; proteostasis; trafficking

DOI:  https://doi.org/10.1016/j.jbc.2022.102423
J Biol Chem. 2022 Aug 25. pii: S0021-9258(22)00867-5. [Epub ahead of print] 102424

Oxidation of two cysteines within yeast Hsp70 impairs proteostasis while directly triggering an Hsf1-dependent cytoprotective response.

Alec Santiago, Kevin A Morano.

  Neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases affect millions of Americans every year. One factor linked to formation of aggregates associated with these diseases is damage sustained to proteins by oxidative stress. Management of protein misfolding by the ubiquitous Hsp70 chaperone family can be modulated by modification of two key cysteines in the ATPase domain by oxidizing or thiol-modifying compounds. To investigate the biological consequences of cysteine modification on the Hsp70 Ssa1 in budding yeast, we generated cysteine null (cysteine to serine) and oxidomimetic (cysteine to aspartic acid) mutant variants of both C264 and C303 and demonstrate reduced ATP binding, hydrolysis, and protein folding properties in both the oxidomimetic and hydrogen peroxide-treated Ssa1. In contrast, cysteine nullification rendered Ssa1 insensitive to oxidative inhibition. Additionally, we determined the oxidomimetic ssa1-2CD (C264D, C303D) allele was unable to function as the sole Ssa1 isoform in yeast cells and also exhibited dominant negative effects on cell growth and viability. Ssa1 binds to and represses Hsf1, the major transcription factor controlling the heat shock response, and we found the oxidomimetic Ssa1 failed to stably interact with Hsf1, resulting in constitutive activation of the heat shock response. Consistent with our in vitro findings, ssa1-2CD cells were compromised for de novo folding, post-stress protein refolding, and in regulated degradation of a model terminally misfolded protein. Together, these findings pinpoint Hsp70 as a key link between oxidative stress and proteostasis, information critical to understanding cytoprotective systems that prevent and manage cellular insults underlying complex disease states.

Keywords:  Hsp70; chaperone; cysteine; proteostasis; reactive oxygen species; redox; thiol modification

DOI:  https://doi.org/10.1016/j.jbc.2022.102424
Cold Spring Harb Perspect Biol. 2022 Aug 30. pii: a041249. [Epub ahead of print]

The Endoplasmic Reticulum and the Fidelity of Nascent Protein Localization.

Michael J McKenna, Sichen Shao.

High-fidelity protein localization is essential to define the identities and functions of different organelles and to maintain cellular homeostasis. Accurate localization of nascent proteins requires specific protein targeting pathways as well as quality control (QC) mechanisms to remove mislocalized proteins. The endoplasmic reticulum (ER) is the first destination for approximately one-third of the eukaryotic proteome and a major site of protein biosynthesis and QC. In mammalian cells, trafficking from the ER provides nascent proteins access to the extracellular space and essentially every cellular membrane and organelle except for mitochondria and possibly peroxisomes. Here, we discuss the biosynthetic mechanisms that deliver nascent proteins to the ER and the QC mechanisms that interface with the ER to correct or degrade mislocalized proteins.

DOI: https://doi.org/10.1101/cshperspect.a041249
Mol Neurodegener. 2022 Sep 02. 17(1): 57

LRP1 is a neuronal receptor for α-synuclein uptake and spread.

Kai Chen, Yuka A Martens, Axel Meneses, Daniel H Ryu, Wenyan Lu, Ana Caroline Raulin, Fuyao Li, Jing Zhao, Yixing Chen, Yunjung Jin, Cynthia Linares, Marshall Goodwin, Yonghe Li, Chia-Chen Liu, Takahisa Kanekiyo, David M Holtzman, Todd E Golde, Guojun Bu, Na Zhao.

   BACKGROUND: The aggregation and spread of α-synuclein (α-Syn) protein and related neuronal toxicity are the key pathological features of Parkinson's disease (PD) and Lewy body dementia (LBD). Studies have shown that pathological species of α-Syn and tau can spread in a prion-like manner between neurons, although these two proteins have distinct pathological roles and contribute to different neurodegenerative diseases. It is reported that the low-density lipoprotein receptor-related protein 1 (LRP1) regulates the spread of tau proteins; however, the molecular regulatory mechanisms of α-Syn uptake and spread, and whether it is also regulated by LRP1, remain poorly understood.
METHODS: We established LRP1 knockout (LRP1-KO) human induced pluripotent stem cells (iPSCs) isogenic lines using a CRISPR/Cas9 strategy and generated iPSC-derived neurons (iPSNs) to test the role of LRP1 in α-Syn uptake. We treated the iPSNs with fluorescently labeled α-Syn protein and measured the internalization of α-Syn using flow cytometry. Three forms of α-Syn species were tested: monomers, oligomers, and pre-formed fibrils (PFFs). To examine whether the lysine residues of α-Syn are involved in LRP1-mediated uptake, we capped the amines of lysines on α-Syn with sulfo-NHS acetate and then measured the internalization. We also tested whether the N-terminus of α-Syn is critical for LRP1-mediated internalization. Lastly, we investigated the role of Lrp1 in regulating α-Syn spread with a neuronal Lrp1 conditional knockout (Lrp1-nKO) mouse model. We generated adeno-associated viruses (AAVs) that allowed for distinguishing the α-Syn expression versus spread and injected them into the hippocampus of six-month-old Lrp1-nKO mice and the littermate wild type (WT) controls. The spread of α-Syn was evaluated three months after the injection.
RESULTS: We found that the uptake of both monomeric and oligomeric α-Syn was significantly reduced in iPSNs with LRP1-KO compared with the WT controls. The uptake of α-Syn PFFs was also inhibited in LRP1-KO iPSNs, albeit to a much lesser extent compared to α-Syn monomers and oligomers. The blocking of lysine residues on α-Syn effectively decreased the uptake of α-Syn in iPSNs and the N-terminus of α-Syn was critical for LRP1-mediated α-Syn uptake. Finally, in the Lrp1-nKO mice, the spread of α-Syn was significantly reduced compared with the WT littermates.
CONCLUSIONS: We identified LRP1 as a key regulator of α-Syn neuronal uptake, as well as an important mediator of α-Syn spread in the brain. This study provides new knowledge on the physiological and pathological role of LRP1 in α-Syn trafficking and pathology, offering insight for the treatment of synucleinopathies.

Keywords:  Human induced pluripotent stem cells; Lewy body dementia; Low-density lipoprotein receptor-related protein 1; Parkinson’s disease; α-Synuclein

DOI:  https://doi.org/10.1186/s13024-022-00560-w
Front Aging Neurosci. 2022 ;14 998136

Editorial: Utility of protein aggregation assays from laboratory to clinical application.

Alison Jane Ellen Green.



Keywords:  PMCA; RT-QuIC; prion disease; seed amplification assays; α-synuclein

DOI:  https://doi.org/10.3389/fnagi.2022.998136
Curr Stem Cell Res Ther. 2022 Aug 31.

The molecular and functional changes of Neural stem cells in Alzheimer's disease: Can they be reinvigorated to conduct Neurogenesis.

Ejlal Abu-El-Rub, Ramada R Khasawneh, Fatimah A Almahasneh, Basma Milad Aloud, Hana M Zegallai.

  Alzheimer's disease (AD) is considered one of the most complicated neurodegenerative disorders, and it is associated with progressive memory loss and remarkable neurocognitive dysfunction that negatively impacts the ability to perform daily living activities. AD accounts for an estimated 60-80% of dementia cases. AD's previously known pathological basis is the deposition of amyloid β (Aβ) aggregates and the formation of neurofibrillary tangles by tau hyperphosphorylation in the cell bodies of neurons that are located in the hippocampus, neocortex, and certain other regions of the cerebral hemispheres and limbic system. The lack of neurotransmitter Acetylcholine and the activation of oxidative stress cascade may also contribute to the pathogenesis of AD. These pathological events can lead to irreversible loss of neuronal networks and the emergence of memory impairment and cognitive dysfunction that can engender an abnormal change in the personality. AD cannot be cured, and to some extent, the prescribed medications can only manage the symptoms associated with this disease. Several studies reported that the regenerative abilities of Neural stem/progenitor cells (NSCs) remarkably declined in AD which disturbed the balancing power to control its progression. Exogenous infusion or endogenous activation of NSCs may be an ultimate solution to restore the neuronal networks in the brain of AD patients and regenerate the damaged areas responsible for memory and cognition. In this mini-review, we will touch upon the fate of NSCs in AD and the utilization of neurogenesis using modified NSCs to restore the cognitive functions in AD.

Keywords:  Alzheimer’s disease; Neural stem cells; Neurogenesis; Neuroinflammation; Pathogenesis; Repair

DOI:  https://doi.org/10.2174/1574888X17666220831105257
PLoS Genet. 2022 Sep 01. 18(9): e1010371

Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development.

Lisa Patricia Deliu, Michael Turingan, Deeshpaul Jadir, Byoungchun Lee, Abhishek Ghosh, Savraj Singh Grewal.

The regulation of ribosome function is a conserved mechanism of growth control. While studies in single cell systems have defined how ribosomes contribute to cell growth, the mechanisms that link ribosome function to organismal growth are less clear. Here we explore this issue using Drosophila Minutes, a class of heterozygous mutants for ribosomal proteins. These animals exhibit a delay in larval development caused by decreased production of the steroid hormone ecdysone, the main regulator of larval maturation. We found that this developmental delay is not caused by decreases in either global ribosome numbers or translation rates. Instead, we show that they are due in part to loss of Rp function specifically in a subset of serotonin (5-HT) neurons that innervate the prothoracic gland to control ecdysone production. We find that these effects do not occur due to altered protein synthesis or proteostasis, but that Minute animals have reduced expression of synaptotagmin, a synaptic vesicle protein, and that the Minute developmental delay can be partially reversed by overexpression of synaptic vesicle proteins in 5-HTergic cells. These results identify a 5-HT cell-specific role for ribosomal function in the neuroendocrine control of animal growth and development.

DOI: https://doi.org/10.1371/journal.pgen.1010371
J Phys Chem B. 2022 Aug 30.

Computationally-Aided Modeling of Hsp70-Client Interactions: Past, Present, and Future.

Erik B Nordquist, Eugenia M Clerico, Jianhan Chen, Lila M Gierasch.

Hsp70 molecular chaperones play central roles in maintaining a healthy cellular proteome. Hsp70s function by binding to short peptide sequences in incompletely folded client proteins, thus preventing them from misfolding and/or aggregating, and in many cases holding them in a state that is competent for subsequent processes like translocation across membranes. There is considerable interest in predicting the sites where Hsp70s may bind their clients, as the ability to do so sheds light on the cellular functions of the chaperone. In addition, the capacity of the Hsp70 chaperone family to bind to a broad array of clients and to identify accessible sequences that enable discrimination of those that are folded from those that are not fully folded, which is essential to their cellular roles, is a fascinating puzzle in molecular recognition. In this article we discuss efforts to harness computational modeling with input from experimental data to develop a predictive understanding of the promiscuous yet selective binding of Hsp70 molecular chaperones to accessible sequences within their client proteins. We trace how an increasing understanding of the complexities of Hsp70-client interactions has led computational modeling to new underlying assumptions and design features. We describe the trend from purely data-driven analysis toward increased reliance on physics-based modeling that deeply integrates structural information and sequence-based functional data with physics-based binding energies. Notably, new experimental insights are adding to our understanding of the molecular origins of "selective promiscuity" in substrate binding by Hsp70 chaperones and challenging the underlying assumptions and design used in earlier predictive models. Taking the new experimental findings together with exciting progress in computational modeling of protein structures leads us to foresee a bright future for a predictive understanding of selective-yet-promiscuous binding exploited by Hsp70 molecular chaperones; the resulting new insights will also apply to substrate binding by other chaperones and by signaling proteins.

DOI: https://doi.org/10.1021/acs.jpcb.2c03806
Nat Neurosci. 2022 Aug 30.

Pathological structural conversion of α-synuclein at the mitochondria induces neuronal toxicity.

Minee L Choi, Alexandre Chappard, Bhanu P Singh, Catherine Maclachlan, Margarida Rodrigues, Evgeniya I Fedotova, Alexey V Berezhnov, Suman De, Christopher J Peddie, Dilan Athauda, Gurvir S Virdi, Weijia Zhang, James R Evans, Anna I Wernick, Zeinab Shadman Zanjani, Plamena R Angelova, Noemi Esteras, Andrey Y Vinokurov, Katie Morris, Kiani Jeacock, Laura Tosatto, Daniel Little, Paul Gissen, David J Clarke, Tilo Kunath, Lucy Collinson, David Klenerman, Andrey Y Abramov, Mathew H Horrocks, Sonia Gandhi.

Aggregation of alpha-synuclein (α-Syn) drives Parkinson's disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)-derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.

DOI: https://doi.org/10.1038/s41593-022-01140-3