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BAG domain

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Last Updated: 02 July 2021

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General | Latest Info

BAG domain

Identifiers
SymbolBAG
PfamPF02179
InterProIPR003103
SMARTBAG
SCOP21hx1 / SCOPe / SUPFAM

Well-study BCL2-associate Athanogene cochaperone 6 plays a central role in membrane protein quality control, with additional links to apoptosis, gene regulation, and immunoregulation. Recent studies demonstrate that Bag6 forms heterotrimeric Bag6 complex with ubiquitin-like 4A and transmembrane domain recognition complex 35 that mediate fates of membrane proteins in tail-anchor protein targeting, mislocalized protein degradation, and endoplasmic reticulum-associate protein degradation. Many roles of the Bag6 complex are likely to center on its ability to bind exposed hydrophobic regions of proteins, such as transmembrane domains. In cytoplasm, Bag6 complex direct substrates either to targeting factors for ER membrane or to ubiquitylation and subsequent proteasomal degradation. Bag6 initially was described as part of a gene cluster that includes human MHC class III on chromosome 6, resulting in its first name, HLA-Bassociated transcript 3. Genomic localization suggests a role in immune response, and this suggestion has been supported by evidence of its roles in Th1 cell survival, natural killer cell cytotoxicity, and MHC class II molecule presentation. The initial Bag6 link to apoptosis was based on its interaction with Reaper, apoptosis-inducing Drosophila protein. Bag6 was not identified in initial searches for functional homologs of BAG1, which identified four additional proteins. Designation of Bag6 as BAG family member come later from limit sequence homology to define BAG domain and apparent heat shock cognate 70-regulating activity found in other BAG family members. In mammals, Bag6 has been shown to be critical in targeting of TA proteins to ER by transmembrane recognition complex pathway, process best understood in equivalent fungal guide entry of tail-anchored proteins pathway. Although Bag6 is missing in fungi, analogous yeast complex contains two proteins, Get4 and Get5 / Mdy2, which are homologs of mammalian proteins TRC35 and Ubl4A, respectively. In yeast, these two proteins form heterotetramer that regulate handoff of TA protein from cochaperone small, glutamine-rich, tetratricopeptide repeat protein 2 to delivery factor Get3. It is expected that mammalian homologs, along with Bag6, play a similar role. Bag6 also interacts with other proteins such as apoptosis-inducing factor, glycoprotein 78, regulatory particle 5, and brother of regulator of imprinted sites and can homo-oligomerize, increasing the level of complexity. These findings build a picture of Bag6 as a central hub for a diverse physiological network of proteins. A variety of diseases, ranging from cancer to autoimmune disorders and diabetes, are linked to Bag6. Despite this demonstrated importance, structural characterization of the Bag6 complex is lacking. The longest and most common isoform of Bag6 gene encodes 1 132-aa protein with a N-terminal ubiquitin-like domain that has been characterized structurally, large proline-rich central domain that is predict to be unstructured, and a C-terminal predict BAG domain. In this study, we mapped TRC35-and Ubl4A-binding regions to the C terminus of Bag6.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

INTRODUCTION

Proteins are complex biological macromolecules that synthesize on ribosomes as linear chains of amino acids that must fold into their native three-dimensional structural conformations to function. Although necessary information required to achieve this functional state is encoded in the primary amino acid sequence, concentrate cellular environment imposes challenge to proper folding, especially during stress. Cells respond to Protein-unfolding stresses such as heat shock by rapidly inducing expression of a wide array of highly conserve cytoprotective genes, including heat shock proteins, also know as molecular chaperones. Molecular chaperones of the Hsp70 class are present in all cells, have high degree of conservation, and are distributed in cytosol, mitochondria, and endoplasmic reticulum. In Saccharomyces cerevisiae, Hsp70 homologs promote protein folding, facilitate translocation across membranes, and provide protection from proteotoxic stresses. In yeast cytosol, two families of Hsp70s, Ssa and Ssb, have been well study. Ssa proteins are involved in protein folding, translation, translocation of proteins into ER and mitochondria, and regulation of heat shock response. Nonessential Ssb chaperones are tether to translating ribosomes through associations with ribosome-associate complex, compose of proteins Ssz1 and Zuo1, supporting dedicated role for Ssb1 / 2 in cotranslational Protein folding. Hsp70s are weak ATPases and share domain architecture consisting of an amino-terminal nucleotide binding domain and carboxyl-terminal substrate binding domain. Hsp70 assists in substrate folding by iterative cycles of ATP binding, hydrolysis, and nucleotide exchange at NBD tightly couple to substrate binding and release at SBD through allosteric mechanism. J-domain-containing cochaperones stimulate ATP hydrolysis, and cofactors such as GrpE in bacteria and BAG-1, HspBP1, or Hsp110 in eukaryotes accelerate the rate of ADP / ATP exchange from NBD, thereby completing the folding cycle. Together, J proteins and NEFs significantly accelerate the ATPase rate of Hsp70 and in turn increase its Protein-folding ability. The BAG-1 Protein was first identified as binding partner of apoptosis inhibitor Protein Bcl-2. It was later shown that conserved region of about 100 amino acids at the carboxyl terminus of BAG-1 interacts directly with NBD of Hsc70 and stimulates ATPase activity in the presence of J Protein. The crystal structure of 13-kDa BAG domain of BAG-1 in complex with ATPase domain of Hsc70 shows monomeric three-helix bundle architecture with helices 2 and 3 making contact with critical residues methionine 61, arginine 261, and glutamate 283 in subdomains IB and IIB of Hsp70 ATPase domain. Binding of BAG domain induces conformational distortion between lobes I and II in NBD, leading to reduced affinity for nucleotides and validating the role of Hsp70 NEF. Alignments of putative BAG domains from different species identify SNL1 as the sole BAG domain-containing Protein in S. Cerevisiae. Previously reported in vitro studies indicate that SNL1 interacts with yeast Ssa and Ssb and regulates ATPase activity of mammalian Hsp70.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

MATERIALS AND METHODS

Growth on rich media of wild type, Bag101, Bag102, Spc7-23 and double mutants was compared at indicated temperatures. Abundance and kinetochore localization of Spc7-23-GFP Protein or, as control, wild type Spc7-GFP, was analyzed in cells at 25C or at 30C for 6 hours. Each fluorescent signal represents Spc7-23-GFP kinetochore signal of individual interphase cell as kinetochores are cluster at the spindle pole body during interphase. Note that at 30C Spc7-23-GFP signal is reduced in wild type or Bag101 background. In Bag102 background Spc7-23-GFP signal is increase at both temperatures. Scale bar: 5 M. Equal or unequal DNA segregation was quantified in Spc7-23-GFP and Spc7-23-GFP Bag102 cells at 30C. * P < 0. 01 for Spc7-23-GFP Bag102 strain compared to Spc7-23-GFP strain. For Spc7-23-GFP and Spc7-23-GFP Bag102 late anaphase cells. Note that wild type DNA segregation is re-establish in Spc7-23bag102 double mutant. Spc7-3HA or Spc7-23-3HA was precipitate using antibodies to HA or control antibodies of the same isotype against irrelevant Protein at either 4C or 30C. Precipitate material was resolved by SDS-PAGE and analyzed by blotting for presence of HSP70. Blotting to HA serves as loading control. Note that Spc7-23 interacts with HSP70, especially at 30C. The Data presented here is compatible with a model where nuclear protein becomes structurally perturb to a degree where it is still functional, but molecular chaperones detect it as being misfolded. Protein is then ubiquitylated by E2 and E3 enzymes Ubc4, Ubr11 and San1, and direct to 26S proteasome via Bag102. Finally, at 26S proteasome, protein is deubiquitylated by DUB Ubp3 and degrade. Ubiquitin is show as grey spheres. Structural perturbation is show as star.


1 Introduction

Parkinsons disease is an incurable neurodegenerative disease which affects 1-2% of the population over the age of 60. PD is characterized by significant loss of dopaminergic neurons within substantia nigra pars compacta as well as presence of Lewy bodies, intracellular inclusions comprise largely of aggregate alpha-synuclein. While exact mechanisms are still unknown, oligomeric species of alpha-synuclein are strongly believed to contribute to cell death observed in PD and other diseases associated with LBs, including dementia with LBs, multiple system atrophy, and Alzheimers disease. Various factors modulate alpha-synuclein processing and aggregation, including molecular chaperones. Chaperones serve to fold nascent proteins, refold misfolded proteins, or direct misfolded proteins for degradation via either the ubiquitin-proteasome system or autophagy lysosome pathway. Heat shock protein 70 is a chaperone which has been shown to be involved in alpha-synuclein processing and preferentially binds to alpha-synuclein fibrils. HSP70 can reduce levels of misfolded and aggregate alpha-synuclein, and protect against alpha-synuclein-mediate toxicity. Co-chaperones are proteins that regulate the function of chaperones by modulating their ATPase activity. One such family of co-chaperones is the BAG family of co-chaperones which act as nucleotide exchange factors that promote ADP release. Bcl-2 associate athanogene co-chaperone family includes six members that are defined by presence of C-terminal BAG domain. The BAG domain consists of three amphipathic alpha helices and ranges from 74 to 112 amino acids in size. This domain is responsible for physical association with HSP70, dimerization with other BAG proteins, and other interactions critical for cell function. BAG co-chaperones not only regulate HSP70 folding activity but also interact with proteins within UPS and ALP, which together facilitate degradation of HSP70 client proteins, and function in other processes including cell division and apoptosis. For example, BAG1 contains ubiquitin-like domain, which interacts with 26S proteasome and thereby facilitates physical link between HSP70 and UPS. BAG1 is also Bcl-2 interacting protein with anti-apoptotic activity. BAG3 interacts with p62, ALP adaptor protein, which promotes proteostasis by facilitating increased protein degradation via ALP as cells age. BAG5 is unique among BAG co-chaperones in that it contains five BAG domains rather than one. BAG5 interacts with HSP70 and inhibits its folding activity. BAG5 also interacts with and inhibits ubiquitin E3 ligase activities of parkin and C-terminal HSP70 interacting protein. Inhibition of HSP70, parkin, and CHIP by BAG5 disrupts proteostasis, mitophagy, and promotes formation of alpha-synuclein oligomers, as well as other protein aggregates, which contribute to neuronal death. Consistent with these findings, BAG5 was found to promote dopaminergic neuron death in substantia nigra in rodent models of PD and interact with other PD relevant proteins including LRRK2, PINK1, and DJ-1.


3 Results and discussion

Using mass spectrometry-base screen for BAG5 interacting proteins, we find that BAG5 interacts with a rich network of chaperones as well as numerous proteins that function within UPS and ALP. Novel HSP70-independent interaction between BAG5 and p62 was identified and verify. These results suggest that BAG5 can interact with ALP in an HSP70-independent manner and may therefore serve as a molecular bridge between the chaperone network and ALP. BAG5 has previously been implicated in autophagy as it forms complex with PD-relevant proteins LRRK2, Rab7L1, and cyclin-Gassociated kinase to promote turnover of trans-Golgi network, activity that impacts ALP function. Interestingly, unlike interaction between BAG5 and p62, association of BAG5 with this complex was at least partially facilitated by HSP70. More recently, we have demonstrated that BAG5 also modulates parkin-dependent mitophagy, suggesting that this co-chaperone may have a more general role in regulating these processes. Further investigation will be required to understand the mechanisms by which this occurs and the role that other BAG proteins may play in these complex intracellular processes required to maintain proteostasis. In addition, while the H4 neuroglioma cell line used in this study is accepted in vitro model for interrogation of PD-relevant molecular pathways, function of BAG5-p62 interaction merits further investigation in vivo. P62 is known to regulate aggregation and degradation of proteins associated with neurodegenerative disease including alpha-synuclein as well as tau, and huntingtin. Moreover, p62 is often co-localize with protein aggregate observed in these neurodegenerative proteinopathies. Therefore, observations that BAG5 interacts with p62, promotes soluble p62 levels, and co-localizes with and supports stability of p62 perinuclear aggresomes, suggest that this interaction may have important implications in disease-associated disturbances of proteostasis. Using luciferase PCA, we find that BAG5 enhances both alpha-synuclein oligomer and p62 protein levels. Considering that p62 has previously been shown to support formation of neuronal protein aggregates, we hypothesize that the effect of BAG5 on alpha-synuclein oligomers was mediated by its effect on p62 protein levels. However, target p62 KD on its own was not sufficient to reduce alpha-synuclein oligomers in our model, although there was significant reduction in alpha-synuclein levels. Therefore, it is likely that the effect of BAG5 on alpha-synuclein oligomerization in this study was mediate through alternative p62-independent pathways. For example, BAG5 is known to inhibit E3 ligase activity of CHIP, which, in turn, inhibits CHIP-mediate proteasomal degradation of alpha-synuclein and enhances alpha-synuclein oligomerization. This could partially explain how BAG5 can enhance alpha-synuclein oligomerization in p62-independent manner. Moreover, numerous proteins involved in UPS and ALP that were identified as putative BAG5 interactors in our proteomic screen indicate that further research is required to dissect complex molecular mechanisms that mediate the effect of BAG5 on alpha-synuclein oligomerization and degradation. Give well-characterize role of p62 in formation and subsequent degradation of protein aggregates, it was surprising that p62 KD alone does not reduce alpha-synuclein oligomers.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

ACKNOWLEDGMENTS

Domain structure of BAG-1 isoforms, of carboxyl terminus of HSC70 interacting protein, and of model for functional cooperation of 2 cochaperones during sorting of HSC70-bound substrate proteins to proteasome for degradation. Four isoforms of BAG-1 have been detect, which differ with regard to the length of their amino termini. Several structural elements are located at the amino terminus, including nuclear localization signal, deoxyribonucleic acid-binding motif, and multiple repeats of hexapeptide motif TRSEEX. All isoforms possess central ubiquitin-like domain use for proteasome binding and carboxyl terminal BAG domain, which mediate binding and regulation of HSC70. CHIP possesses triple TPR domain that, together with adjacent charge region, forms the binding site for HSC70 and Hsp90. The Central coil coil domain may be involved in protein-protein interactions. The Carboxyl terminal U-box is required for ubiquitin ligase activity of CHIP and seems to mediate binding to ubiquitin-conjugating enzymes of the Ubc4 / 5 family. The Association of CHIP with carboxyl terminal domain of HSC70 gave rise to the chaperone complex that mediates sorting to proteasome. At the same time, BAG-1 binds to the adenosine triphosphatase domain of HSC70 and in addition contacts CHIP directly. The Ubiquitin-like domain of BAG-1 remains exposed in form complex and serves as proteasomal sorting signal. Subunit and domain arrangement in complex remains to be determined experimentally

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Sources

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

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