NEDDylation promotes stress granule assembly

Jayabalan, Aravinth Kumar; Sanchez, Anthony; Park, Ra Young; Yoon, Sang Pil; Kang, Gum–Yong; Baek, Je‐Hyun; Anderson, Paul; Kee, Younghoon; Ohn, Takbum

doi:10.1038/ncomms12125

Cited by 71 publications

(72 citation statements)

References 53 publications

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“…It has been suggested that G3BP has both antiviral and proviral activities by activating PKR (95,96) and suppressing P1234 translation (97), that it escorts the incoming genomic RNA to the plasma membrane (37), and that it regulates the switch from translation to transcription of genomic RNA (91). G3BP1 is the target for several posttranslational modifications (e.g., phosphorylation, ADP ribosylation, acetylation, methylation, NEDDylation, and ubiquitylation) that regulate its endonuclease activity and its participation in the formation of stress granules and other protein complexes (94,(98)(99)(100)(101)(102)(103)(104)(105)(106)(107). The presence or absence and the location of these modifications likely also play a regulatory role in interaction with nsP3 and genomic RNA for initiation of alphavirus replication.…”

Section: Discussionmentioning

confidence: 99%

ADP-ribosyl–binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication

Abraham

Hauer

McPherson

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

111

139

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Alphaviruses are plus-strand RNA viruses that cause encephalitis, rash, and arthritis. The nonstructural protein (nsP) precursor polyprotein is translated from genomic RNA and processed into four nsPs. nsP3 has a highly conserved macrodomain (MD) that binds ADP-ribose (ADPr), which can be conjugated to protein as a posttranslational modification involving transfer of ADPr from NAD + by poly ADPr polymerases (PARPs). The nsP3 MD also removes ADPr from mono ADP-ribosylated (MARylated) substrates. To determine which aspects of alphavirus replication require nsP3 MD ADPr-binding and/or hydrolysis function, we studied NSC34 neuronal cells infected with chikungunya virus (CHIKV). Infection induced ADP-ribosylation of cellular proteins without increasing PARP expression, and inhibition of MARylation decreased virus replication. CHIKV with a G32S mutation that reduced ADPr-binding and hydrolase activities was less efficient than WT CHIKV in establishing infection and in producing nsPs, dsRNA, viral RNA, and infectious virus. CHIKV with a Y114A mutation that increased ADPr binding but reduced hydrolase activity, established infection like WT CHIKV, rapidly induced nsP translation, and shut off host protein synthesis with reduced amplification of dsRNA. To assess replicase function independent of virus infection, a transreplicase system was used. Mutant nsP3 MD s D10A, G32E, and G112E with no binding or hydrolase activity had no replicase activity, G32S had little, and Y114A was intermediate to WT. Therefore, ADP ribosylation of proteins and nsP3 MD ADPr binding are necessary for initiation of alphavirus replication, while hydrolase activity facilitates amplification of replication complexes. These observations are consistent with observed nsP3 MD conservation and limited tolerance for mutation. alphavirus | macrodomain | ADP ribosylation | replication complexes | PARP

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Section: Discussionmentioning

confidence: 99%

ADP-ribosyl–binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication

Abraham

Hauer

McPherson

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

111

139

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“…However, our results also clearly establish that acute inhibition of active protein ubiquitylation or neddylation does not directly impact SG formation or dissolution despite the large impact on overall protein ubiquitylation. Previous studies demonstrated that long-term (18hr) NAE inhibition or knockdown of neddylation components inhibits SG formation 20, 21 . Near complete NAE inhibition using the N8-E1i takes place on minute timescales when added to mammalian cells 45 .…”

Section: Discussionmentioning

confidence: 99%

Active protein neddylation or ubiquitylation is dispensable for stress granule dynamics

Markmiller

Fulzele

Higgins

et al. 2018

Preprint

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32Many protein homeostasis stressors induce the formation of membraneless cytoplasmic stress 33 granules (SGs) that contain large assemblies of repressed mRNAs and associated RNA binding 34 proteins. Similar stressors have been shown to globally alter the function of the ubiquitin 35 proteasome system (UPS) resulting in the accumulation of ubiquitylated proteins. Previous 36 studies have demonstrated that ubiquitin and specific UPS components co-localize with SGs and 37 that reducing the abundance or activity of ubiquitin pathway proteins can inhibit SG formation. 38These studies suggest that SG dynamics and composition may be regulated by ubiquitylation of 39 SG resident proteins. Using ubiquitin-specific proteomic approaches, we demonstrate that many 40 proteins, including some SG proteins are dynamically ubiquitylated upon SG-inducing sodium 41 arsenite treatment. We utilized potent and selective inhibitors of the ubiquitin activating enzyme 42 (UAE) or the NEDD8 activating enzyme (NAE) to directly test if active protein ubiquitylation or 43 neddylation was required for SG dynamics. Using ubiquitin-site specific proteomics, we establish 44 that UAE inhibition results in the rapid loss of nearly all protein ubiquitylation regardless of 45 ubiquitin chain type. Addition of UAE or NAE inhibitors to cells did not alter arsenite-induced SG 46 formation or dissolution. While we confirmed that ubiquitin co-localizes with both sodium arsenite 47 and thapsigargin-induced SGs, antibodies that recognize all forms of ubiquitin more strongly co-48 localize with SGs compared to antibodies that preferentially recognize polyubiquitin or specific 49 polyubiquitin-linkages. Interestingly, ubiquitin itself co-localizes with SGs in a UAE independent 50 manner suggesting that the ubiquitin present within SGs is likely unconjugated ubiquitin. Our 51 findings clearly demonstrate that active protein ubiquitylation or neddylation is not required for SG 52 dynamics. These results suggest that ubiquitin-binding SG proteins may recruit free ubiquitin into 53SGs to modulate SG protein interactions. 54 55 56 57 58 59 60 61 62 63 64 65

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“…NEDD8 (neural precursor cell‐expressed developmentally downregulated protein 8) is a small, ubiquitin‐like protein that covalently attaches to protein substrates in a manner similar to ubiquitination, known as neddylation. Neddylation of serine/arginine (SR)‐rich splicing factor 3 (SRSF3), an SG regulator, is necessary for SG formation (Jayabalan et al, 2016). Reversible protein acetylation is another important protein PTM modulated by histone acetylases and histone deacetylases.…”

Section: Stress Granule Formation and Regulationmentioning

confidence: 99%

The involvement of stress granules in aging and aging‐associated diseases

2020

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Stress granules (SGs) are nonmembrane assemblies formed in cells in response to stress conditions. SGs mainly contain untranslated mRNA and a variety of proteins. RNAs and scaffold proteins with intrinsically disordered regions or RNA‐binding domains are essential for the assembly of SGs, and multivalent macromolecular interactions among these components are thought to be the driving forces for SG assembly. The SG assembly process includes regulation through post‐translational modification and involvement of the cytoskeletal system. During aging, many intracellular bioprocesses become disrupted by factors such as cellular environmental changes, mitochondrial dysfunction, and decline in the protein quality control system. Such changes could lead to the formation of aberrant SGs, as well as alterations in their maintenance, disassembly, and clearance. These aberrant SGs might in turn promote aging and aging‐associated diseases. In this paper, we first review the latest progress on the molecular mechanisms underlying SG assembly and SG functioning under stress conditions. Then, we provide a detailed discussion of the relevance of SGs to aging and aging‐associated diseases.

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NEDDylation promotes stress granule assembly

Cited by 71 publications

References 53 publications

ADP-ribosyl–binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication

ADP-ribosyl–binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication

Active protein neddylation or ubiquitylation is dispensable for stress granule dynamics

The involvement of stress granules in aging and aging‐associated diseases

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