Biallelic Mutations in TBCD , Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy

Flex, Elisabetta; Niceta, Marcello; Cecchetti, Serena; Thiffault, Isabelle; Au, Margaret G.; Capuano, Alessandro; Piermarini, Emanuela; Ivanova, Anna; Francis, Joshua W.; Chillemi, Giovanni; Chandramouli, Balasubramanian; Carpentieri, Giovanna; Haaxma, Charlotte A.; Ciolfi, Andrea; Pizzi, Simone; Douglas, Ganka; Levine, Kara L.; Sferra, Antonella; Dentici, Maria Lisa; Pfundt, Rolph; Pichon, Jean Baptiste Le; Farrow, Emily; Baas, Frank; Piemonte, Fiorella; Dallapiccola, Bruno; Graham, John M.; Saunders, Carol J.; Bertini, Enrico; Kahn, Richard; Koolen, David A.; Tartaglia, Marco

doi:10.1016/j.ajhg.2016.08.003

Cited by 68 publications

(99 citation statements)

References 67 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As a consequence, we propose the TBCD⅐ARL2⅐␤-tubulin complex to be a key player in the folding process and potentially separately in regulating the lifetime or stability of the microtubule array. Finally, the studies by Flex et al (17) and others (18,19,20,39) highlight the clinical significance of the TBCD⅐ARL2⅐␤-tubulin trimer and suggest that continued study of this complex will prove to be beneficial to both the scientific and clinical communities.…”

Section: Discussionmentioning

confidence: 99%

“…When a subset of the identified TBCD mutants were co-expressed with ARL2 in our HEK cell system, they result in a reduced amount of ␤-tubulin in the TBCD⅐ARL2⅐␤-tubulin complex. Additionally, the purified complexes each have reduced stability (lower melting point, T m ) in a thermal denaturation assay (17). The study also analyzed patient cells in an in vivo tubulin polymerization (nocodazole washout) assay.…”

Section: Discussionmentioning

confidence: 99%

“…More recently, however, TBCD has been implicated in additional cellular roles, including actions at centrosomes (12)(13)(14) and at the cell surface (15,16). Several recent studies have identified a number of point mutations in TBCD found in patients with links to early-onset encephalopathy (17)(18)(19) and intractable seizures (20). Throughout the time when roles for TBCD in microtubule biology were being identified, there have also been strong functional links to the ARL2 GTPase, a member of the ADP-ribosylation factor (ARF) family of regulatory GTPases.…”

mentioning

confidence: 99%

See 2 more Smart Citations

A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and β-Tubulin Is Required for Maintaining the Microtubule Network

Francis

Newman

Cunningham

et al. 2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Edited by Velia M. FowlerMicrotubule dynamics involves the polymerization and depolymerization of tubulin dimers and is an essential and highly regulated process required for cell viability, architecture, and division. The regulation of the microtubule network also depends on the maintenance of a pool of ␣␤-tubulin heterodimers. These dimers are the end result of complex folding and assembly events, requiring the TCP1 Ring Complex (TriC or CCT) chaperonin and five tubulin-specific chaperones, tubulin binding cofactors A-E (TBCA-TBCE). However, models of the actions of these chaperones are incomplete or inconsistent. We previously purified TBCD from bovine tissues and showed that it tightly binds the small GTPase ARL2 but appears to be inactive. Here, in an effort to identify the functional form of TBCD and using non-denaturing gels and immunoblotting, we analyzed lysates from a number of mouse tissues and cell lines to identify the quaternary state(s) of TBCD and ARL2. We found that both proteins co-migrated in native gels in a complex of ϳ200 kDa that also contained ␤-tubulin. Using human embryonic kidney cells enabled the purification of the TBCD⅐ARL2⅐␤-tubulin trimer found in cell and tissue lysates as well as two other novel TBCD complexes. Characterization of ARL2 point mutants that disrupt binding to TBCD suggested that the ARL2-TBCD interaction is critical for proper maintenance of microtubule densities in cells. We conclude that the TBCD⅐ARL2⅐␤-tubulin trimer represents a functional complex whose activity is fundamental to microtubule dynamics.Microtubules are highly dynamic polymers that are best known for their roles as the central cytoskeletal structure in cells and in mitotic spindles during cell division. They are also the tracks on which organelles traffic, particularly important in polarized cells that generate great distances between parts of the cell. Additionally, they are the core of sensory and motile cilia or flagella. The formation and destruction of microtubules and microtubule bundles are orchestrated by a large number of proteins that include the microtubule-associated proteins. Microtubules are polymers of the ␣␤-tubulin dimer, with several genes encoding each ␣-or ␤-tubulin subunit (e.g. see Lewis et al. (1)), resulting in some diversity in composition. Tubulins can also be modified by posttranslational modifications, including acetylation, tyrosination, and phosphorylation, which can alter the dynamics of the polymerization and depolymerization reactions. Because of the essential role of microtubules in cell division, they have also been the target of many antitumor therapies, e.g. the taxanes and Vinca alkaloids (2). However, despite their importance to cells and in the clinic and decades of research, we still lack a complete molecular-level understanding of the biosynthesis and regulation of the formation of ␣␤-tubulin.Tubulins are typically the most abundant proteins in mammalian cells, but the generation of the ␣␤-tubulin dimer requires a complex series of biosynthetic steps to ...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and β-Tubulin Is Required for Maintaining the Microtubule Network

Francis

Newman

Cunningham

et al. 2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Also, a spate of recent reports describe point mutations in TBCD found in patients with links to early-onset encephalopathy [28–31] and intractable seizures [32]. A subset of these mutations were specifically shown to disrupt formation and stability of the TBCD•ARL2•β-tubulin complex [29], which we have recently purified and characterized [11]. …”

Section: Introductionmentioning

confidence: 99%

Nucleotide Binding to ARL2 in the TBCD ∙ ARL2 ∙ β-Tubulin Complex Drives Conformational Changes in β-Tubulin

Francis

Goswami

Novick

et al. 2017

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

Microtubules are highly dynamic tubulin polymers that are required for a variety of cellular functions. Despite the importance of a cellular population of tubulin dimers, we have incomplete information about the mechanisms involved in the biogenesis of αβ-tubulin heterodimers. In addition to prefoldin and the TCP-1 Ring Complex, five tubulin-specific chaperones, termed cofactors A-E (TBCA-E), and GTP are required for the folding of α- and β-tubulin subunits and assembly into heterodimers. We recently described the purification of a novel trimer, TBCD•ARL2•β-tubulin. Here, we employed hydrogen/deuterium exchange coupled with mass spectrometry to explore the dynamics of each of the proteins in the trimer. Addition of guanine nucleotides resulted in changes in the solvent accessibility of regions of each protein that led to predictions about each’s role in tubulin folding. Initial testing of that model confirmed that it is ARL2, and not β-tubulin, that exchanges GTP in the trimer. Comparisons of the dynamics of ARL2 monomer to ARL2 in the trimer suggested that its protein interactions were comparable to those of a canonical GTPase with an effector. This was supported by the use of nucleotide binding assays that revealed an increase in the affinity for GTP by ARL2 in the trimer. We conclude that the TBCD•ARL2•β-tubulin complex represents a functional intermediate in the β-tubulin folding pathway whose activity is regulated by the cycling of nucleotides on ARL2. The co-purification of guanine nucleotide on the β-tubulin in the trimer is also shown, with implications to modeling the pathway.

show abstract

“…Somatic single-nucleotide variants were detected using Mutect software v.1.1.6 [40] and small indels were identified through a comparison between indels called in individual C-CSC lines and their matched nontumoral samples by means of the GATK Haplotype Caller algorithm [41], as previously described [42, 43]. The resulting SNVs and small indels were annotated by SnpEff v3.6 [44] and dbNSFP2.8 [45] in terms of functional impact of variants [46, 47]. Variant validation and genotyping were performed by direct sequencing using the ABI BigDye Terminator Sequencing kit (Applied Biosystems) and an ABI3500 capillary sequencer (Applied Biosystems).…”

Section: Methodsmentioning

confidence: 99%

HIPK2-T566 autophosphorylation diversely contributes to UV- and doxorubicin-induced HIPK2 activation

et al. 2017

Self Cite

View full text Add to dashboard Cite

HIPK2 is a Y-regulated S/T kinase involved in various cellular processes, including cell-fate decision during development and DNA damage response. Cis-autophosphorylation in the activation-loop and trans-autophosphorylation at several S/T sites along the protein are required for HIPK2 activation, subcellular localization, and subsequent posttranslational modifications. The specific function of a few of these autophosphorylations has been recently clarified; however, most of the sites found phosphorylated by mass spectrometry in human and/or mouse HIPK2 are still uncharacterized. In the process of studying HIPK2 in human colorectal cancers, we identified a mutation (T566P) in a site we previously found autophosphorylated in mouse Hipk2. Biochemical and functional characterization of this site showed that compared to wild type (wt) HIPK2, HIPK2-T566P maintains nuclear-speckle localization and has only a mild reduction in kinase and growth arresting activities upon overexpression. Next, we assessed cell response following UV-irradiation or treatment with doxorubicin, two well-known HIPK2 activators, by evaluating cell number and viability, p53-Ser46 phosphorylation, p21 induction, and caspase cleavage. Interestingly, cells expressing HIPK2-T566P mutant did not respond to UV-irradiation, while behaved similarly to wt HIPK2 upon doxorubicin-treatment. Evaluation of HIPK2-T566 phosphorylation status by a T566-phospho-specific antibody showed constitutive phosphorylation in unstressed cells, which was maintained after doxorubicin-treatment but inhibited by UV-irradiation. Taken together, these data show that HIPK2-T566 phosphorylation contributes to UV-induced HIPK2 activity but it is dispensable for doxorubicin response.

show abstract

Biallelic Mutations in TBCD , Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy

Cited by 68 publications

References 67 publications

A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and β-Tubulin Is Required for Maintaining the Microtubule Network

A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and β-Tubulin Is Required for Maintaining the Microtubule Network

Nucleotide Binding to ARL2 in the TBCD ∙ ARL2 ∙ β-Tubulin Complex Drives Conformational Changes in β-Tubulin

HIPK2-T566 autophosphorylation diversely contributes to UV- and doxorubicin-induced HIPK2 activation

Contact Info

Product

Resources

About