Structural Insights into KCTD Protein Assembly and Cullin3 Recognition

Ji, Alan X.; Chu, Anh; Nielsen, T.K.; Benlekbir, Samir; Rubinstein, John L.; Privé, Gilbert G.

doi:10.1016/j.jmb.2015.08.019

Cited by 54 publications

(116 citation statements)

References 55 publications

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“…Thereby, our data add for the first time ZBTB38 to the short list of BTB-domain proteins that were recently shown to not bind to cullin-3 [59, 60]. …”

Section: Discussionmentioning

confidence: 87%

Cullin E3 Ligase Activity Is Required for Myoblast Differentiation

Blondelle

Shapiro

Domenighetti

et al. 2017

Journal of Molecular Biology

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The role of cullin E3-ubiquitin ligases for muscle homeostasis is best known during muscle atrophy, as the cullin-1 substrate adaptor atrogin-1 counts among the most well-characterized muscle atrogins. We investigated, whether cullin activity was also crucial during terminal myoblast differentiation and aggregation of acetylcholine receptors for the establishment of neuromuscular junctions in vitro. The activity of cullin E3-ligases is modulated through posttranslational modification with the small ubiquitin-like modifier nedd8. Using either the Nae1 inhibitor MLN4924 (Pevonedistat) or siRNA against nedd8 in early or late stages of differentiation on C2C12 myoblasts, and primary satellite cells from mouse and human, we show that cullin E3-ligase activity is necessary for each step of the muscle cell differentiation program in vitro. We further investigate known transcriptional repressors for terminal muscle differentiation ZBTB38, Bhlhe41 and Id1. Due to their identified roles for terminal muscle differentiation, we hypothesize that accumulation of these potential cullin E3-ligase substrates may be partially responsible for the observed phenotype. MLN4924 is currently undergoing clinical trials in cancer patients, and our experiments highlight concerns on the homeostasis and regenerative capacity of muscles in these patients who often experience cachexia.

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“…Thereby, our data add for the first time ZBTB38 to the short list of BTB-domain proteins that were recently shown to not bind to cullin-3 [59, 60]. …”

Section: Discussionmentioning

confidence: 87%

Cullin E3 Ligase Activity Is Required for Myoblast Differentiation

Blondelle

Shapiro

Domenighetti

et al. 2017

Journal of Molecular Biology

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“…Solved structures are available for the N‐terminal BTB domain of several KCTD family proteins primarily revealing pentameric homo‐oligomers. This fivefold symmetry appears to extend through the C‐terminus . The BTB domains of KCTD proteins also mediate other protein‐protein interactions, leading to three main hypotheses for general KCTD mechanisms.…”

Section: Structure and Function Of Kctd Family Proteinsmentioning

confidence: 99%

“…The distinguishing feature of KCTD proteins is a single N‐terminal BTB/POZ (bric‐a‐brac, tramtrak, and broad complex/poxvirus zinc finger) domain, the exception being KCTD19 with three separate BTBs that may reflect tandem gene amplification (Figure ) . BLAST searches readily reveal that the BTB domains of KCTD family proteins are most similar in amino acid sequence to the T1/BTB domains that mediate tetramerization of voltage‐gated potassium channel subunits to form functional channels . This sequence similarity to Kv channels explains how KCTDs acquired their official name (potassium channel tetramerization domain).…”

Section: Introductionmentioning

confidence: 99%

“…This feature is consistent with their proposed roles as adaptor molecules that use their C‐termini to bind and recruit diverse cellular proteins destined for degradation. In this model, KCTDs are responsible for selecting protein substrates for ubiquitination by cullin‐RING ubiquitin ligases (CRLs) that bind to the BTB domains of KCTD proteins . Thus, disrupted proteostasis required for the delicate balance between protein function versus degradation could potentially underlie neurological disorders now associated with the brain‐enriched proteins KCTD3, KCTD7, KCTD13, and KCTD17.…”

Section: Introductionmentioning

confidence: 99%

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KCTD: A new gene family involved in neurodevelopmental and neuropsychiatric disorders

et al. 2019

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The underlying molecular basis for neurodevelopmental or neuropsychiatric disorders is not known. In contrast, mechanistic understanding of other brain disorders including neurodegeneration has advanced considerably. Yet, these do not approach the knowledge accrued for many cancers with precision therapeutics acting on well‐characterized targets. Although the identification of genes responsible for neurodevelopmental and neuropsychiatric disorders remains a major obstacle, the few causally associated genes are ripe for discovery by focusing efforts to dissect their mechanisms. Here, we make a case for delving into mechanisms of the poorly characterized human KCTD gene family. Varying levels of evidence support their roles in neurocognitive disorders ( KCTD3 ), neurodevelopmental disease ( KCTD7 ), bipolar disorder ( KCTD12 ), autism and schizophrenia ( KCTD13 ), movement disorders ( KCTD17 ), cancer ( KCTD11 ), and obesity ( KCTD15 ). Collective knowledge about these genes adds enhanced value, and critical insights into potential disease mechanisms have come from unexpected sources. Translation of basic research on the KCTD‐related yeast protein Whi2 has revealed roles in nutrient signaling to mTORC1 (KCTD11) and an autophagy‐lysosome pathway affecting mitochondria (KCTD7). Recent biochemical and structure‐based studies (KCTD12, KCTD13, KCTD16) reveal mechanisms of regulating membrane channel activities through modulation of distinct GTPases. We explore how these seemingly varied functions may be disease related.

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“…Despite its name, KCTD17 has been shown to act as a substrate-adaptor for Cul3-RING ubiquitin ligases (CRL3s) to regulate proteosomal degradation of specific targets. 22, 36, 37 We observed that hepatic Kctd17 expression is robustly increased in HFD-fed and db/db mice (Figure 4A). Next, we confirmed our LC-MS/MS results by immunoprecipitation of endogenous KCTD17 in livers from HFD-fed mice with anti-PHLPP2 antibody, and vice versa (Figure 4B).…”

Section: Resultsmentioning

confidence: 88%

Degradation of PHLPP2 by KCTD17, via a Glucagon-Dependent Pathway, Promotes Hepatic Steatosis

et al. 2017

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BACKGROUND & AIMS Obesity-induced non-alcoholic fatty liver disease (NAFLD) develops, in part, via excess insulin-stimulated hepatic de novo lipogenesis, which increases, paradoxically, in patients with obesity-induced insulin resistance. Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) terminates insulin signaling by dephosphorylating Akt; levels of PHLPP2 are reduced in livers from obese mice. We investigated whether loss of hepatic PHLPP2 is sufficient to induce fatty liver in mice, mechanisms of PHLPP2 degradation in fatty liver, and expression of genes that regulate PHLPP2 in livers of patients with NAFLD. METHODS C57BL/6J mice (controls), obese db/db mice and mice with liver-specific deletion of PHLPP2 (L-PHLPP2) fed either normal chow or high-fat diet (HFD) were analyzed for metabolic phenotypes including glucose tolerance and hepatic steatosis. PHLPP2-deficient primary hepatocytes or CRISPR/Cas9-mediated PHLPP2-knockout hepatoma cells were analyzed for insulin signaling and gene expression. We performed mass spectrometry analyses of livers tissues from C57BL/6J mice transduced with Ad-HA-FLAG-PHLPP2 to identify post-translational modifications to PHLPP2 and proteins that interact with PHLPP2. We measured levels of mRNAs by quantitative reverse transcription PCR in liver biopsies from patients with varying degrees of hepatic steatosis. RESULTS PHLPP2-knockout hepatoma cells and hepatocytes from L-PHLPP2 mice showed normal initiation of insulin signaling, but prolonged insulin action. Chow-fed L-PHLPP2 mice had normal glucose tolerance but hepatic steatosis. In HFD-fed C57BL/6J or db/db obese mice, endogenous PHLPP2 was degraded by glucagon and PKA-dependent phosphorylation of PHLPP2 (at Ser1119 and Ser1210), which led to PHLPP2 binding to potassium channel tetramerization domain containing 17 (KCTD17), a substrate-adaptor for Cul3-RING ubiquitin ligases. Levels of KCTD17 mRNA were increased in livers of HFD-fed C57BL/6J or db/db obese mice and in liver biopsies patients with NAFLD, compared with liver tissues from healthy control mice or patients without steatosis. Knockdown of KCTD17 with small hairpin RNA in primary hepatocytes increased PHLPP2 protein but not Phlpp2 mRNA, indicating that KCTD17 mediates PHLPP2 degradation. KCTD17 knockdown in obese mice prevented PHLPP2 degradation and decreased expression of lipogenic genes. CONCLUSIONS In mouse models of obesity, we found that PHLPP2 degradation induced lipogenesis without affecting gluconeogenesis. KCTD17, which is upregulated in liver tissues of obese mice and patients with NAFLD, binds to phosphorylated PHLPP2 to target it for ubiquitin-mediated degradation; this increases expression of genes that regulate lipogenesis to promote hepatic steatosis. Inhibitors of this pathway might be developed for treatment of patients with NAFLD.

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Structural Insights into KCTD Protein Assembly and Cullin3 Recognition

Cited by 54 publications

References 55 publications

Cullin E3 Ligase Activity Is Required for Myoblast Differentiation

Cullin E3 Ligase Activity Is Required for Myoblast Differentiation

KCTD: A new gene family involved in neurodevelopmental and neuropsychiatric disorders

Degradation of PHLPP2 by KCTD17, via a Glucagon-Dependent Pathway, Promotes Hepatic Steatosis

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