Dynein is the primary minus-end-directed microtubule motor protein. To achieve activation, dynein binds to the dynactin complex and an adaptor to form the "activated dynein complex." The protein Lis1 aids activation by binding to dynein and promoting its association with dynactin and the adaptor. Ndel1 and its paralog Nde1 are dynein- and Lis1-binding proteins that help control dynein localization within the cell. Cell-based assays suggest that Ndel1-Nde1 also work with Lis1 to promote dynein activation, although the underlying mechanism is unclear. Using purified proteins and quantitative binding assays, here we found that the C-terminal region of Ndel1 contributes to dynein binding and negatively regulates binding to Lis1. Using single-molecule imaging and protein biochemistry, we observed that Ndel1 inhibits dynein activation in two distinct ways. First, Ndel1 disfavors the formation of the activated dynein complex. We found that phosphomimetic mutations in the C-terminal domain of Ndel1 increase its ability to inhibit dynein-dynactin-adaptor complex formation. Second, we observed that Ndel1 interacts with dynein and Lis1 simultaneously and sequesters Lis1 away from its dynein-binding site. In doing this, Ndel1 prevents Lis1-mediated dynein activation. Together, our work suggests that in vitro, Ndel1 is a negative regulator of dynein activation, which contrasts with cellular studies where Ndel1 promotes dynein activity. To reconcile our findings with previous work, we posit that Ndel1 functions to scaffold dynein and Lis1 together while keeping dynein in an inhibited state. We speculate that Ndel1 release can be triggered in cellular settings to allow for timed dynein activation.
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Zinc controls PML nuclear body formation through regulation of a paralog specific auto-inhibition in SUMO1
SUMO proteins are important regulators of many key cellular functions in part through their ability to form interactions with other proteins containing SUMO interacting motifs (SIMs). One characteristic feature of all SUMO proteins is the presence of a highly divergent intrinsically disordered region at their N-terminus. In this study, we examine the role of this N-terminal region of SUMO proteins in SUMO–SIM interactions required for the formation of nuclear bodies by the promyelocytic leukemia (PML) protein (PML-NBs). We demonstrate that the N-terminal region of SUMO1 functions in a paralog specific manner as an auto-inhibition domain by blocking its binding to the phosphorylated SIMs of PML and Daxx. Interestingly, we find that this auto-inhibition in SUMO1 is relieved by zinc, and structurally show that zinc stabilizes the complex between SUMO1 and a phospho-mimetic form of the SIM of PML. In addition, we demonstrate that increasing cellular zinc levels enhances PML-NB formation in senescent cells. Taken together, these results provide important insights into a paralog specific function of SUMO1, and suggest that zinc levels could play a crucial role in regulating SUMO1-SIM interactions required for PML-NB formation and function.
more » « less- NSF-PAR ID:
- 10369265
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 50
- Issue:
- 14
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- p. 8331-8348
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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ABSTRACT EseN is an
Edwardsiella ictaluri type III secretion system effector with phosphothreonine lyase activity. In this work, we demonstrate that EseN inactivates p38 and c-Jun-N-terminal kinase (JNK) in infected head-kidney-derived macrophages (HKDMs). We have previously reported inactivation of extracellular-regulated kinase 1/2 (ERK1/2). Also, for the first time, we demonstrated that EseN is involved in the inactivation of 3-phosphoinositide-dependent kinase 1 (PDK1), which has not been previously demonstrated for any of the EseN homologs in other species. We also found that EseN significantly affected mRNA expression ofIL-10 , pro-apoptoticbaxa , andp53 , but had no significant effect on anti-apoptoticbcl2 or pro-apoptotic apoptotic peptidase activating factor 1. EseN is also involved in the inhibition of caspase-8 and caspase-3/7 but does not affect caspase-9 activity. Repression of apoptosis was further confirmed with flow cytometry using Alexa Fluor 647-labeled annexin V and propidium iodide. In addition, we found that theE. ictaluri T3SS is essential for the inhibition of IL-1β maturation, but EseN is not involved in this process. EseN did not affect cell pyroptosis, as indicated by the lack of EseN impact on the release of lactate dehydrogenase from infected HKDM. The transmission electron microscopy data also indicate that HKDM infected with WT or aneseN mutant died by apoptosis, while HKDM infected with the T3SS mutant more likely died by pyroptosis. Collectively, our results indicate thatE. ictaluri EseN is involved in inactivation of ERK1/2, p38, JNK, and PDK1 signaling pathways that lead to modulation of cell death among infected HKDMs.IMPORTANCE This work has global significance in the catfish industry, which provides food for increasing global populations.
E. ictaluri is a leading cause of disease loss, and EseN is an important player inE. ictaluri virulence. TheE. ictaluri T3SS effector EseN plays an essential role in establishing infection, but the specific role EseN plays is not well characterized. EseN belongs to a family of phosphothreonine lyase effectors that specifically target host mitogen activated protein kinase (MAPK) pathways important in regulating host responses to infection. No phosphothreonine lyase equivalents are known in eukaryotes, making this family of effectors an attractive target for indirect narrow-spectrum antibiotics. Targeting of major vault protein and PDK1 kinase by EseN has not been reported in EseN homologs in other pathogens and may indicate unique functions ofE. ictaluri EseN. EseN targeting of PDK1 is particularly interesting in that it is linked to an extraordinarily diverse group of cellular functions. -
Babitzke, Paul (Ed.)ABSTRACT Oxidative stress causes cellular damage, including DNA mutations, protein dysfunction, and loss of membrane integrity. Here, we discovered that a TrmB (transcription regulator of mal operon) family protein (Pfam PF01978) composed of a single winged-helix DNA binding domain (InterPro IPR002831) can function as thiol-based transcriptional regulator of oxidative stress response. Using the archaeon Haloferax volcanii as a model system, we demonstrate that the TrmB-like OxsR is important for recovery of cells from hypochlorite stress. OxsR is shown to bind specific regions of genomic DNA, particularly during hypochlorite stress. OxsR-bound intergenic regions were found proximal to oxidative stress operons, including genes associated with thiol relay and low molecular weight thiol biosynthesis. Further analysis of a subset of these sites revealed OxsR to function during hypochlorite stress as a transcriptional activator and repressor. OxsR was shown to require a conserved cysteine (C24) for function and to use a CG-rich motif upstream of conserved BRE/TATA box promoter elements for transcriptional activation. Protein modeling suggested the C24 is located at a homodimer interface formed by antiparallel α helices, and that oxidation of this cysteine would result in the formation of an intersubunit disulfide bond. This covalent linkage may promote stabilization of an OxsR homodimer with the enhanced DNA binding properties observed in the presence of hypochlorite stress. The phylogenetic distribution TrmB family proteins, like OxsR, that have a single winged-helix DNA binding domain and conserved cysteine residue suggests this type of redox signaling mechanism is widespread in Archaea. IMPORTANCE TrmB-like proteins, while not yet associated with redox stress, are found in bacteria and widespread in archaea. Here, we expand annotation of a large group of TrmB-like single winged-helix DNA binding domain proteins from diverse archaea to function as thiol-based transcriptional regulators of oxidative stress response. Using Haloferax volcanii as a model, we reveal that the TrmB-like OxsR functions during hypochlorite stress as a transcriptional activator and repressor of an extensive gene coexpression network associated with thiol relay and other related activities. A conserved cysteine residue of OxsR serves as the thiol-based sensor for this function and likely forms an intersubunit disulfide bond during hypochlorite stress that stabilizes a homodimeric configuration with enhanced DNA binding properties. A CG-rich DNA motif in the promoter region of a subset of sites identified to be OxsR-bound is required for regulation; however, not all sites have this motif, suggesting added complexity to the regulatory network.more » « less
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