A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

Pirone, Lucía; Xolalpa, Wendy; Sigurðsson, Jón Otti; Ramírez, Juanma; Pérez, Coralia; González, Monika; Sabando, Ainara Ruiz de; Elortza, Félix; Rodríguez, Manuel Sánchez; Mayor, Ugo; Olsen, Jesper V.; Barrio, Rosa; Sutherland, James D.

doi:10.1038/srep40756

Cited by 61 publications

(63 citation statements)

References 89 publications

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“…All together, these results show that LUZP1 levels are sensitive to degradation via the UPS pathway and suggest that truncated SALL1 may contribute to this process. Furthermore, we compared LUZP1 ubiquitination in 293 WT vs 293 335 cells using the BioUb strategy (see Materials and Methods) (44). We could observe a prominent band in presence of BioUb, likely corresponding to the monoubiquitinated form of LUZP1 in the pulldowns (Figure 7G).…”

Section: Resultsmentioning

confidence: 99%

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LUZP1, a novel regulator of primary cilia and the actin cytoskeleton, is altered in Townes-Brocks Syndrome

Bozal-Basterra

Gonzalez-Santamarta

Bermejo-Arteagabeitia

et al. 2019

Preprint

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36Primary cilia are sensory organelles that are crucial for cell signaling during 37 development and organ homeostasis. Cilia arise from the centrosome and their 38 formation is governed by numerous regulatory factors. We show that the leucine-zipper 39 protein LUZP1 localizes to the pericentriolar material and actin cytoskeleton. Using 40TurboID proximity labeling and pulldowns, LUZP1 associates with factors linked to 41 centrosome and actin filaments. Loss of LUZP1 reduces F-actin levels, facilitating 42 ciliogenesis and altering Sonic Hedgehog signaling, pointing to a key role in the 43 cytoskeleton-cilia interdependency. Moreover, we show that LUZP1 interacts with a 44 truncated form of the transcription factor SALL1 that causes Townes-Brocks Syndrome. 45 TBS is characterized by digit, heart and kidney malformations and is linked in part to 46 defective cilia. Truncated SALL1 increases the ubiquitin proteasome-mediated 47 51Primary cilia are sensory organelles that have a crucial role in cell signaling, 52 polarity and protein trafficking during development and organ homeostasis. 53 Importantly, the involvement of primary cilia in the above-mentioned processes is 54 frequently due to its role in Sonic Hedgehog (Shh) pathway regulation (1). Briefly, Shh 55 activation through its receptor PTCH1 leads to ciliary enrichment of the transmembrane 56 protein Smoothened (SMO), with concomitant conversion of the transcription factor 57 GLI3 from a cleaved repressor form to a full-length activator form, leading to activation 58 of Shh target genes. Two such genes are PTCH1 and GLI1 (encoding the Shh receptor 59 and a transcriptional activator, respectively), exemplifying the feedback and fine-tuning 60 of the Shh pathway. 61Cilia arise from the centrosome, a cellular organelle composed of two barrel-62 shaped microtubule-based structures called the centrioles. Primary cilia formation is 63 very dynamic throughout the cell cycle. Cilia are nucleated from the mother centriole 64 (MC) at the membrane-anchored basal body upon entry into the G0 phase, and they 65 reabsorb as cells progress from G1 to S phase, completely disassembling in mitosis (2). 66Centrioles are surrounded by protein-based matrix pericentriolar material (PCM) (3, 4). 67In eukaryotic cells, PCM proteins are concentrically arranged around a centriole in a 68 highly organized manner (5-8). Based on this observation, proper positioning and 69 organization of PCM proteins may be important for promoting different cellular 70 processes in a spatially regulated way (9). Not surprisingly, aberrations in the function 71 of PCM scaffolds are also associated with many human diseases, including cancer and 72 ciliopathies (10, 11). 73Cilia assembly and disassembly are regulated by diverse factors, including the 74 main cilia suppressor proteins CCP110 and CEP97 and the actin cytoskeleton. CCP110 75 4 and CEP97 form a complex that, when removed from the MC, allows ciliogenesis (12). 76The regulation of actin dynamics is also considered a major ciliogenesis driver in 7...

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Section: Resultsmentioning

confidence: 99%

“…All constructs were verified by Sanger sequencing. Plasmids CAG-BioUBC(x4)_BirA_V5_puro (called here BioUb) and CAG-BirA-puro (called here BirA) were reported previously (44).…”

Section: Methodsmentioning

confidence: 99%

LUZP1, a novel regulator of primary cilia and the actin cytoskeleton, is altered in Townes-Brocks Syndrome

Bozal-Basterra

Gonzalez-Santamarta

Bermejo-Arteagabeitia

et al. 2019

Preprint

Self Cite

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“…57,68 In addition, previous proteomic analyses indicated hundreds of potential substrates. 67,69,70 Therefore, it is essential to identify key ufmylated substrates that are involved in the timing of progression to mitosis, which will greatly expand our knowledge of this UBL and facilitate a better understanding of the exact molecular pathogenesis of ufmylation-related microcephaly. 67,69,70 Therefore, it is essential to identify key ufmylated substrates that are involved in the timing of progression to mitosis, which will greatly expand our knowledge of this UBL and facilitate a better understanding of the exact molecular pathogenesis of ufmylation-related microcephaly.…”

Section: Discussionmentioning

confidence: 99%

The UFM1 cascade times mitosis entry associated with microcephaly

Deng

et al. 2019

The FASEB Journal

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Posttranslational modifications enhance the functional diversity of the proteome by modifying the substrates. The UFM1 cascade is a novel ubiquitin‐like modification system. The mutations in UFM1, its E1 (UBA5) and E2 (UFC1), have been identified in patients with microcephaly. However, its pathological mechanisms remain unclear. Herein, we observed the disruption of the UFM1 cascade in Drosophila neuroblasts (NBs) decreased the number of NBs, leading to a smaller brain size. The lack of ufmylation in NBs resulted in an increased mitotic index and an extended G2/M phase, indicating a defect in mitotic progression. In addition, live imaging of the embryos revealed an impaired E3 ligase (Ufl1) function resulted in premature entry into mitosis and failed cellularization. Even worse, the embryonic lethality occurred as early as within the first few mitotic cycles following the depletion of Ufm1. Knockdown of ufmylation in the fixed embryos exhibited severe phenotypes, including detached centrosomes, defective microtubules, and DNA bridge. Furthermore, we observed that the UFM1 cascade could alter the level of phosphorylation on tyrosine‐15 of CDK1 (pY15‐CDK1), which is a negative regulator of the G2 to M transition. These findings yield unambiguous evidence suggesting that the UFM1 cascade is a microcephaly‐causing factor that regulates the progression of the cell cycle at mitosis phase entry.

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“…Ubiquitin fold modifier 1 (UFM1) is a ubiquitin-like protein (UBL) that has a role in several cellular processes, including unfolded-protein response and erythroid development (1)(2)(3)(4)(5)(6)(7). Recently, it has been shown that prevention of that modification leads to severe anemia that, in mice, is lethal to embryos (8).…”

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confidence: 99%

Trans ‐binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding

Mashahreh¹,

Hassouna²,

Soudah³

et al. 2018

FASEB j.

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All ubiquitin-like proteins (UBLs) undergo an activation process before their conjugation to target proteins. Although the steps required for the activation of UBLs are conserved and common to all UBLs, we have previously shown that the activation of the UBL, ubiquitin fold modifier 1 (UFM1) by the E1, Ufm1 modifier-activating enzyme 5 (UBA5) is executed in a trans-binding mechanism, not observed in any other E1. In this study, we explored the necessity of that mechanism for UFM1 activation and found that it is needed not only for UFM1 binding to UBA5 but also for stabilizing the UBA5 homodimer. Although UBA5 functions as a dimer, in solution it behaves as a weak dimer. Dimerization of UBA5 is required for ATP binding; therefore, stabilization of the dimer by UFM1 enhances ATP binding. Our results make a connection between the binding of UFM1 to UBA5 and the latter's affinity to ATP, so we propose a novel mechanism for the regulation of ATP's binding to E1.-Mashahreh, B., Hassouna, F., Soudah, N., Cohen-Kfir, E., Strulovich, R., Haitin, Y., Wiener, R. Trans-binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding.

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A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

Cited by 61 publications

References 89 publications

LUZP1, a novel regulator of primary cilia and the actin cytoskeleton, is altered in Townes-Brocks Syndrome

LUZP1, a novel regulator of primary cilia and the actin cytoskeleton, is altered in Townes-Brocks Syndrome

The UFM1 cascade times mitosis entry associated with microcephaly

Trans ‐binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding

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