Abstract Dermal fibroblasts and cutaneous nerves are important players in skin diseases, while their reciprocal roles during skin inflammation have not been characterized. Here we identify an inflammation-induced subset of papillary fibroblasts that promotes aberrant neurite outgrowth and psoriasiform skin inflammation by secreting the extracellular matrix protein tenascin-C (TNC). Single-cell analysis of fibroblast lineages reveals a Tnc + papillary fibroblast subset with pro-axonogenesis and neuro-regulation transcriptomic hallmarks. TNC overexpression in fibroblasts boosts neurite outgrowth in co-cultured neurons, while fibroblast-specific TNC ablation suppresses hyperinnervation and alleviates skin inflammation in male mice modeling psoriasis. Dermal γδT cells, the main producers of type 17 pathogenic cytokines, frequently contact nerve fibers in mouse psoriasiform lesions and are likely modulated by postsynaptic signals. Overall, our results highlight the role of an inflammation-responsive fibroblast subset in facilitating neuro-immune synapse formation and suggest potential avenues for future therapeutic research.
In the present study, we hypothesized that hypoxia-inducible factor 1α (HIF-1α)-mediated mitophagy plays a protective role in ischemia/reperfusion (I/R)-induced acute kidney injury (AKI). Mitophagy was evaluated by measuring the changes of mitophagy flux, mitochondria DNA copy number, and the changes of mitophagy-related proteins including translocase of outer mitochondrial membrane 20 (TOMM20), cytochrome c oxidase IV (COX IV), microtubule-associated protein 1 light chain 3B (LC3B), and mitochondria adaptor nucleoporin p62 in HK2 cells, a human tubular cell line. Results show that HIF-1α knockout significantly attenuated hypoxia/reoxygenation (H/R)-induced mitophagy, aggravated H/R-induced apoptosis, and increased the production of reactive oxygen species (ROS). Similarly, H/R induced significantly increase in Bcl-2 19-kDa interacting protein 3 (BNIP3), a downstream regulator of HIF-1α. Notably, BNIP3 overexpression reversed the inhibitory effect of HIF-1α knockout on H/R-induced mitophagy, and prevented the enhancing effect of HIF-1α knockout on H/R-induced apoptosis and ROS production. For in vivo study, we established HIF-1αflox/flox; cadherin-16-cre mice in which tubular HIF-1α was specifically knockout. It was found that tubular HIF-1α knockout significantly inhibited I/R-induced mitophagy, and aggravated I/R-induced tubular apoptosis and kidney damage. In contrast, adenovirus-mediated BNIP3 overexpression significantly reversed the decreased mitophagy, and prevented enhanced kidney damage in tubular HIF-1α knockout mice with I/R injury. In summary, our study demonstrated that HIF-1α-BNIP3-mediated mitophagy in tubular cells plays a protective role through inhibition of apoptosis and ROS production in acute kidney damage.
Abstract Background Brown adipocytes (BAs) are major components of brown adipose tissue (BAT), which is involved in blood pressure regulation. BAs are derived from multiple progenitors, including PDGFRα + adipose-derived stem cells (ASCs). Skin-derived mesenchymal stem cells (S-MSCs) have the capacity to differentiate into adipocytes; however, their ability to differentiate into BAs remains unexplored. We aim to study the ability and regulatory mechanism of the differentiation of S-MSCs into BAs and the direct role of BAT in blood pressure regulation. Methods Protein expression was measured by flow cytometry or Western blotting, and gene mRNA levels were quantified by real-time quantitative PCR (RT-PCR). To induce the differentiation of S-MSCs into BAs, S-MSCs were stimulated with a brown adipogenic cocktail comprising insulin, IBMX, dexamethasone, triiodothyronine (T3), and rosiglitazone for the indicated periods. The oxygen consumption rate (OCR) was measured with an XF24 Extracellular Flux Analyzer. Mitochondrial mass was determined by flow cytometry and fluorescence staining. Hypertension was induced in WT mice by infusion of angiotensin II (Ang II), and systolic blood pressure (SBP) was measured using a tail cuff. Interscapular brown adipose tissue (iBAT)-deficient mice were generated by surgical removal of the iBAT depot, after which the animals were allowed to recover for 6 days. Aortic, iBAT, and heart tissue sections were analyzed by hematoxylin and eosin (HE) staining. Results We found that in vitro, S-MSCs isolated from the mouse dermis expressed the stem cell markers CD90/105 and PDGFRα and readily differentiated into BAs. Mitochondrial biogenesis and oxygen consumption were markedly increased during differentiation of S-MSCs into BAs . In vivo, iBAT was converted to white adipose tissue (WAT) in Ang II-induced hypertensive mice. We assessed the direct role of BAT in blood pressure (BP) regulation by using iBAT-deficient mice (generated by surgical removal of iBAT) and C57BL/6 (wild-type (WT)) mice and found that Ang II-induced BP elevation and vascular damage were markedly aggravated in iBAT-deficient mice compared with WT mice. Conclusions This study demonstrates that PDGFRα + S-MSCs are able to differentiate into BAs and that this differentiation is regulated by mitochondrial activity. We also show that BAT plays a direct role in ameliorating Ang II-induced hypertension. The therapeutic potential of BAT for the prevention of hypertension-induced organ remodeling thus warrants further investigation. Graphical abstract. Schematic of the in vitro differentiation of PDGFRα + S-MSCs into BAs via a process regulated by mitochondrial activity. BAT plays a direct role in Ang II-induced hypertension and target organ remodeling
Abstract Psoriasis is a common inflammatory skin disorder with no cure. Mesenchymal stem cells (MSCs) have immunomodulatory properties for psoriasis, but the therapeutic efficacies varied, and the molecular mechanisms were unknown. In this study, we improved the efficacy by enhancing the immunomodulatory effects of umbilical cord–derived MSCs (UC-MSCs). UC-MSCs stimulated by TNF-α and IFN-γ exhibited a better therapeutic effect in a mouse model of psoriasis. Single-cell RNA sequencing revealed that the stimulated UC-MSCs overrepresented a subpopulation expressing high tryptophanyl-tRNA synthetase 1 (WARS1). WARS1-overexpressed UC-MSCs treat psoriasis-like skin inflammation more efficiently than control UC-MSCs by restraining the proinflammatory macrophages. Mechanistically, WARS1 maintained a RhoA-Akt axis and governed the immunomodulatory properties of UC-MSCs. Together, we identify WARS1 as a master regulator of UC-MSCs with enhanced immunomodulatory capacities, which paves the way for the directed modification of UC-MSCs for escalated therapeutic efficacy.
Mammalian brain tubulins undergo a reversible posttranslational modification—polyglutamylation—which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins, both with preference for the β-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for α-isoform than β-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. Tandem mass spectrometry analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl termini of α1- and β2-tubulins and a recombinant tubulin. Particularly, in the recombinant α1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules premodified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by TTLL4-initiated sites. Finally, we showed that kinesin behaves differentially on microtubules modified by two enzymes. This study underpins the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and sheds light on their distinct role in vivo. Mammalian brain tubulins undergo a reversible posttranslational modification—polyglutamylation—which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins, both with preference for the β-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for α-isoform than β-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. Tandem mass spectrometry analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl termini of α1- and β2-tubulins and a recombinant tubulin. Particularly, in the recombinant α1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules premodified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by TTLL4-initiated sites. Finally, we showed that kinesin behaves differentially on microtubules modified by two enzymes. This study underpins the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and sheds light on their distinct role in vivo. Polyglutamylation is a reversible protein post-translational modification, by which proteins are attached with a side chain of glutamate residues. First, the γ-carboxyl of a glutamate residue in the primary structure of protein is linked to a free glutamate through an amide bond to form the branch point. Variable number of glutamate residues are then sequentially added mainly through α-carboxyl linkage to form a side chain (1Janke C. Magiera M.M. The tubulin code and its role in controlling microtubule properties and functions.Nat. Rev. Mol. Cell Biol. 2020; 21: 307-326Crossref PubMed Scopus (304) Google Scholar). Members of the tubulin tyrosine ligase like (TTLL) family, TTLL1, 4, 5, 6, 7, 9, 11, and 13, have been demonstrated as authentic glutamate ligases that differentially catalyze the formation of the γ- (initiation) or α-linkage (elongation) (2van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. A targeted multienzyme mechanism for selective microtubule polyglutamylation.Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 3Kubo T. Yanagisawa H.-A. Yagi T. Hirono M. Kamiya R. Tubulin polyglutamylation regulates axonemal motility by modulating activities of inner-arm dyneins.Curr. Biol. 2010; 20: 441-445Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Conversely, the shortening and removal of polyglutamate side chains are catalyzed by the 6-member cytosolic carboxypeptidase (CCP) family. Except CCP5 that specifically catalyzes removal of the branch point γ-carboxyl linked monoglutamate, the other CCPs all involve in shortening the side chain through hydrolysis of the α-carboxyl-linked glutamate (4Rogowski K. van Dijk J. Magiera M.M. Bosc C. Deloulme J.-C. 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Role of cytosolic carboxypeptidase 5 in neuronal survival and spermatogenesis.Sci. Rep. 2017; 7: 41428Crossref PubMed Scopus (19) Google Scholar). The homeostasis of polyglutamylation is of importance in neuronal survival (4Rogowski K. van Dijk J. Magiera M.M. Bosc C. Deloulme J.-C. Bosson A. et al.A family of protein-deglutamylating enzymes associated with neurodegeneration.Cell. 2010; 143: 564-578Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 9Fernandez-Gonzalez A. Purkinje cell degeneration (pcd) phenotypes caused by mutations in the axotomy-induced gene, Nna1.Science. 2002; 295: 1904-1906Crossref PubMed Scopus (199) Google Scholar, 10Magiera M.M. Bodakuntla S. Žiak J. Lacomme S. Marques Sousa P. Leboucher S. et al.Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport.EMBO J. 2018; 37: 2313Crossref Scopus (83) Google Scholar, 11Berezniuk I. Vu H.T. Lyons P.J. Sironi J.J. Xiao H. 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Xiong Z. et al.Glutamylation of the DNA sensor cGAS regulates its binding and synthase activity in antiviral immunity.Nat. Immunol. 2016; 17: 369-378Crossref PubMed Scopus (148) Google Scholar). The substrate specificity of TTLL glutamylases was mainly determined using tubulin, the best-known substrate of polyglutamylation. The C-terminal intrinsically disordered regions of α- and β-tubulins exposed on the outer surface of microtubule (MT) are the hotspots for polyglutamylation (15Garnham C.P. Roll-Mecak A. The chemical complexity of cellular microtubules: tubulin post-translational modification enzymes and their roles in tuning microtubule functions.Cytoskeleton (Hoboken). 2012; 69: 442-463Crossref PubMed Scopus (128) Google Scholar, 16Redeker V. Mass spectrometry analysis of C-terminal posttranslational modifications of tubulins.Methods Cell Biol. 2010; 95: 77-103Crossref PubMed Scopus (52) Google Scholar). With tubulins from mammalian brain or non-neuronal cell lines as the substrates, TTLL glutamylases were found not only distinguishable for their activities in initiation or elongation but also for their preference for α-tubulin or β-tubulin (2van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. A targeted multienzyme mechanism for selective microtubule polyglutamylation.Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). For instance, TTLL4 and TTLL5 were characterized as initiators that prefer β-tubulin and α-tubulin respectively (2van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. A targeted multienzyme mechanism for selective microtubule polyglutamylation.Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). TTLL1, 6, 11, and 13 all seemingly prefer to the elongation on α-tubulin, whereas TTLL7 catalyzes both initiation and elongation on β-tubulin (2van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. A targeted multienzyme mechanism for selective microtubule polyglutamylation.Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 17Mukai M. Ikegami K. Sugiura Y. Takeshita K. Nakagawa A. Setou M. Recombinant mammalian tubulin polyglutamylase TTLL7 performs both initiation and elongation of polyglutamylation on β-tubulin through a random sequential pathway.Biochemistry. 2009; 48: 1084-1093Crossref PubMed Scopus (32) Google Scholar). In addition, TTLL4 is known as an exclusive initiator (18Mahalingan K.K. Keenen E.K. Strickland M. Li Y. Liu Y. Ball H.L. et al.Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes.Nat. Struct. Mol. Biol. 2020; 27: 802-813Crossref PubMed Scopus (23) Google Scholar) and able to ligate glutamate to a broad range of proteins (19Van Dijk J. Miro J. Strub J.M. Lacroix B. van Dorsselaer A. Edde B. et al.Polyglutamylation is a post-translational modification with a broad range of substrates.J. Biol. Chem. 2008; 283: 3915-3922Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), such as nucleoplasmin, PELP, NAP1, and NAP2 (20Kashiwaya K. Nakagawa H. Hosokawa M. Mochizuki Y. Ueda K. Piao L. et al.Involvement of the tubulin tyrosine ligase-like family member 4 polyglutamylase in PELP1 polyglutamylation and chromatin remodeling in pancreatic cancer cells.Cancer Res. 2010; 70: 4024-4033Crossref PubMed Scopus (37) Google Scholar, 21Regnard C. Polyglutamylation of nucleosome assembly proteins.J. Biol. Chem. 2000; 275: 15969-15976Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 22Onikubo T. Nicklay J.J. Xing L. Warren C. Anson B. Wang W.-L. et al.Developmentally regulated post-translational modification of nucleoplasmin controls histone sequestration and deposition.Cell Rep. 2015; 10: 1735-1748Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Mouse brain tubulins are barely glutamylated at birth, and the polyglutamylation mainly takes place during postnatal development, with α-tubulin being glutamylated first. By the time of adult, the majority of brain tubulin is polyglutamylated (23Audebert S. Koulakoff A. Berwald-Netter Y. Gros F. Denoulet P. Edde B. Developmental regulation of polyglutamylated alpha- and beta-tubulin in mouse brain neurons.J. Cell Sci. 1994; 107: 2313-2322Crossref PubMed Google Scholar). TTLL1 and TTLL7 are the major brain glutamylases, and knocking out either can reduce the tubulin glutamylation level in the brain (12Wu H.-Y. Rong Y. Bansal P.K. Wei P. Guo H. Morgan J.I. TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice.PLoS Genet. 2022; 18e1010144Crossref Scopus (3) Google Scholar, 24Ikegami K. Heier R.L. Taruishi M. Takagi H. Mukai M. Shimma S. et al.Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3213-3218Crossref PubMed Scopus (185) Google Scholar, 25Janke C. Rogowski K. Wloga D. Regnard C. Kajava A.V. Strub J.-M. et al.Tubulin polyglutamylase enzymes are members of the TTL domain protein family.Science. 2005; 308: 1758-1762Crossref PubMed Scopus (243) Google Scholar, 26Bodakuntla S. Yuan X. Genova M. Gadadhar S. Leboucher S. Birling M.-C. et al.Distinct roles of α- and β-tubulin polyglutamylation in controlling axonal transport and in neurodegeneration.EMBO J. 2021; 40e108498Crossref PubMed Scopus (20) Google Scholar, 27Ikegami K. Mukai M. Tsuchida J.-I. Heier R.L. Macgregor G.R. Setou M. TTLL7 is a mammalian beta-tubulin polyglutamylase required for growth of MAP2-positive neurites.J. Biol. Chem. 2006; 281: 30707-30716Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In contrary, the prototypic CCP—Nna1/CCP1 is the most efficient eraser that shortens the side chain and contributes substantially to the polyglutamylation homeostasis in the nervous system (4Rogowski K. van Dijk J. Magiera M.M. Bosc C. Deloulme J.-C. Bosson A. et al.A family of protein-deglutamylating enzymes associated with neurodegeneration.Cell. 2010; 143: 564-578Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 9Fernandez-Gonzalez A. Purkinje cell degeneration (pcd) phenotypes caused by mutations in the axotomy-induced gene, Nna1.Science. 2002; 295: 1904-1906Crossref PubMed Scopus (199) Google Scholar, 28Wu H.-Y. Rong Y. Correia K. Min J. Morgan J.I. Comparison of the enzymatic and functional properties of three cytosolic carboxypeptidase family members.J. Biol. Chem. 2015; 290: 1222-1232Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Polyglutamylation homeostasis is required for neuronal survival. Most prominently, loss of Nna1/CCP1 causes severe neurodegeneration across species (9Fernandez-Gonzalez A. Purkinje cell degeneration (pcd) phenotypes caused by mutations in the axotomy-induced gene, Nna1.Science. 2002; 295: 1904-1906Crossref PubMed Scopus (199) Google Scholar, 29Zhao X. Onteru S.K. Dittmer K.E. Parton K. Blair H.T. Rothschild M.F. et al.A missense mutation in AGTPBP1 was identified in sheep with a lower motor neuron disease.Heredity (Edinb). 2012; 109: 156-162Crossref PubMed Scopus (24) Google Scholar, 30Karakaya M. Paketci C. Altmueller J. Thiele H. Hoelker I. Yis U. et al.Biallelic variant in AGTPBP1causes infantile lower motor neuron degeneration and cerebellar atrophy.Am. J. Med. Genet. 2019; 179: 1580-1584PubMed Google Scholar, 31Shashi V. Magiera M.M. Klein D. Zaki M. Schoch K. Rudnik-Schöneborn S. et al.Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration.EMBO J. 2018; 37: 247Crossref Scopus (63) Google Scholar). In the Nna1/CCP1 mutant—Purkinje cell degeneration (pcd) mice, the tubulin polyglutamylation level in both cerebellum and cerebral cortex is elevated (4Rogowski K. van Dijk J. Magiera M.M. Bosc C. Deloulme J.-C. Bosson A. et al.A family of protein-deglutamylating enzymes associated with neurodegeneration.Cell. 2010; 143: 564-578Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar), accompanied by rapid loss of cerebellar Purkinje cells at the time of wean and progressive degeneration of selective neurons in olfactory bulb, retina, and thalamus over about a year (9Fernandez-Gonzalez A. Purkinje cell degeneration (pcd) phenotypes caused by mutations in the axotomy-induced gene, Nna1.Science. 2002; 295: 1904-1906Crossref PubMed Scopus (199) Google Scholar, 32Mullen R.J. Eicher E.M. Sidman R.L. Purkinje cell degeneration, a new neurological mutation in the mouse.Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 208-212Crossref PubMed Scopus (462) Google Scholar). Nna1 mutations also cause tetraplegia in newborn sheep (29Zhao X. Onteru S.K. Dittmer K.E. Parton K. Blair H.T. Rothschild M.F. et al.A missense mutation in AGTPBP1 was identified in sheep with a lower motor neuron disease.Heredity (Edinb). 2012; 109: 156-162Crossref PubMed Scopus (24) Google Scholar) and damaging early-onset neurodegeneration in humans (29Zhao X. Onteru S.K. Dittmer K.E. Parton K. Blair H.T. Rothschild M.F. et al.A missense mutation in AGTPBP1 was identified in sheep with a lower motor neuron disease.Heredity (Edinb). 2012; 109: 156-162Crossref PubMed Scopus (24) Google Scholar, 30Karakaya M. Paketci C. Altmueller J. Thiele H. Hoelker I. Yis U. et al.Biallelic variant in AGTPBP1causes infantile lower motor neuron degeneration and cerebellar atrophy.Am. J. Med. Genet. 2019; 179: 1580-1584PubMed Google Scholar, 31Shashi V. Magiera M.M. Klein D. Zaki M. Schoch K. Rudnik-Schöneborn S. et al.Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration.EMBO J. 2018; 37: 247Crossref Scopus (63) Google Scholar, 33Bosch Grau M. Masson C. Gadadhar S. Rocha C. Tort O. Marques Sousa P. et al.Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration.J. Cell Sci. 2017; 130: 938-949Crossref PubMed Scopus (44) Google Scholar). Accumulating evidence demonstrated that restoring the polyglutamylation homeostasis in pcd mice can prevent neuronal death. Knocking out TTLL1 could rescue the Purkinje cell loss in pcd mice (10Magiera M.M. Bodakuntla S. Žiak J. Lacomme S. Marques Sousa P. Leboucher S. et al.Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport.EMBO J. 2018; 37: 2313Crossref Scopus (83) Google Scholar, 11Berezniuk I. Vu H.T. Lyons P.J. Sironi J.J. Xiao H. Burd B. et al.Cytosolic carboxypeptidase 1 is involved in processing α- and β-tubulin.J. Biol. Chem. 2012; 287: 6503-6517Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12Wu H.-Y. Rong Y. Bansal P.K. Wei P. Guo H. Morgan J.I. TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice.PLoS Genet. 2022; 18e1010144Crossref Scopus (3) Google Scholar), accompanied by a dramatic reduction in cerebellar tubulin glutamylation. Recently, we further identified that TTLL4, a β-tubulin favorable initiator, but not TTLL5, 7, or 11, also contributes to the neuronal loss in pcd mice. Deletion of TTLL4 did not significantly alter the tubulin glutamylation level in cerebellum but rescued the Purkinje cell death in pcd mice (12Wu H.-Y. Rong Y. Bansal P.K. Wei P. Guo H. Morgan J.I. TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice.PLoS Genet. 2022; 18e1010144Crossref Scopus (3) Google Scholar). Notably, although similar to TTLL4, TTLL7 also favors to modify β-tubulin, its loss did not rescue the neuronal degeneration in pcd mice, despite the correction of tubulin glutamylation level in cerebellum (12Wu H.-Y. Rong Y. Bansal P.K. Wei P. Guo H. Morgan J.I. TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice.PLoS Genet. 2022; 18e1010144Crossref Scopus (3) Google Scholar). This raises the question whether TTLL4 and TTLL7 behave similarly even for their common substrate—tubulin. Previous studies showed that recombinant mammalian TTLL7 can equally modify α- and β-tubulins of free form but exhibits higher activity for MT with a preference for β-tubulin (17Mukai M. Ikegami K. Sugiura Y. Takeshita K. Nakagawa A. Setou M. Recombinant mammalian tubulin polyglutamylase TTLL7 performs both initiation and elongation of polyglutamylation on β-tubulin through a random sequential pathway.Biochemistry. 2009; 48: 1084-1093Crossref PubMed Scopus (32) Google Scholar). The mechanism underlying this difference was revealed by analyzing a cryo-EM structure of TTLL7 in complex with MT (34Garnham C.P. Vemu A. Wilson-Kubalek E.M. Yu I. Szyk A. Lander G.C. et al.Multivalent microtubule recognition by tubulin tyrosine ligase-like family glutamylases.Cell. 2015; 161: 1112-1123Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). However, despite its substrate versatility and contribution to neurodegeneration in pcd mice, the properties of TTLL4 have been less studied. In this study, we purified a recombinant mouse TTLL4 and characterized its enzyme properties in a pure in vitro system. Using an antibody-based method, we found that recombinant TTLL4 and TTLL7 exhibit distinct activity and isoform selectivity for tubulins from postnatal mouse brain. Furthermore, with synthetic peptides, which mimics the tails of α1- and β2-tubulins, the most abundant brain isoforms (35Rüdiger M. Plessman U. Klöppel K.D. Wehland J. Weber K. Class II tubulin, the major brain beta tubulin isotype is polyglutamylated on glutamic acid residue 435.FEBS Lett. 1992; 308: 101-105Crossref PubMed Scopus (120) Google Scholar), as the substrates, we show that TTLL4 and TTLL7 exhibit different site selectivity. Their different modification sites were further confirmed with purified recombinant mouse α1A/β2A tubulin dimers (36Diao L. Liu M.-Y. Song Y.-L. Zhang X. Liang X. Bao L. α1A and α1C form microtubules to display distinct properties mainly mediated by their C-terminal tails.J. Mol. Cell Biol. 2022; 13: 864-875Crossref PubMed Scopus (5) Google Scholar). Interestingly, we identified a novel region in the recombinant α1A tubulin that is subjected to glutamylation by two enzymes, still at distinct sites. Finally, we showed that kinesin exhibits very different motility and affinity on MTs modified by TTLL4 and TTLL7. This study underpins the different reactivity and site specificity of TTLL4 and TTLL7 for brain tubulins, and sheds light on their distinct functions in vivo. In order to characterize the enzyme properties of TTLL4, a truncated version of mouse TTLL4, which does not alter the tubulin isoform preference of the enzyme (2van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. A targeted multienzyme mechanism for selective microtubule polyglutamylation.Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), was fused with a histidine-maltose binding protein-tobacco etch virus tag at its N terminus (Fig. 1A) and expressed using a bacterial system. Upon Coomassie brilliant blue (CBB) staining, the major band of purified protein was visualized at the predicated molecular weight (117.9 kDa) after SDS-PAGE, which was also immunoreactive with a His-tag antibody (Fig. 1B). Several antibodies are available to study polyglutamylated proteins. Amongst them, GT335 is able to detect the branch point, including both monomodifications and polymodifications (37Wolff A. de Néchaud B. Chillet D. Mazarguil H. Desbruyères E. Audebert S. et al.Distribution of glutamylated alpha and beta-tubulin in mouse tissues using a specific monoclonal antibody, GT335.Eur. J. Cell Biol. 1992; 59: 425-432PubMed Google Scholar), whereas other antibodies such as B3 and polyE only recognize tubulin side chains with more than two or three glutamate residues, respectively (Fig. 1C) (2van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. A targeted multienzyme mechanism for selective microtubule polyglutamylation.Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 24Ikegami K. Heier R.L. Taruishi M. Takagi H. Mukai M. Shimma S. et al.Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3213-3218Crossref PubMed Scopus (185) Google Scholar, 38Gagnon C. White D. Cosson J. Huitorel P. Edde B. Desbruyères E. et al.The polyglutamylated lateral chain of alpha-tubulin plays a key role in flagellar motility.J. Cell Sci. 1996; 109: 1545-1553Crossref PubMed Google Scholar). To determine the activity of TTLL4, the recombinant enzyme was first incubated with porcine brain tubulin, and the glutamylation level was monitored using GT335 or polyE antibody, with denatured enzyme incubated at the identical condition as control. The porcine tubulin alone was immunoreactive for both GT335 and polyE antibodies, and the signals were not further increased with addition of the recombinant TTLL4 (Fig. S1B). We speculated that the high basal level of glutamylation already existed in this tubulin preparation might have overwhelmed the change in glutamylation level upon recombinant TTLL4 addition. Therefore, tubulins purified from newborn mice brain (NB), which are known little glutamylated, were used to test the activity of the recombinant TTLL4. Indeed, glutamylation levels of tubulin from this resource were much lower compared with those from porcine brain as evidenced by much weaker GT335 immunoreactivity (Figs. 1D and S1B). In reactions containing the intact enzyme, the GT335 signal was obviously increased compared with those with denatured enzyme or without enzyme (Fig. 1D). In contrast, the polyE signal remained unchanged, suggesting its inability to produce longer chain modification (Fig. S1C). Therefore, the recombinant TTLL4 is enzymatically active, and its activity to initiate the glutamylation of NB tubulin is readily detectable using the GT335 antibody. With tubulins purified from NB as the substrate, we sought to characterize the enzyme properties of the recombinant TTLL4 using an immunoblotting-based method. The pH dependency of this enzyme was analyzed in a range between pH 5.5 and 9.0. The enzyme activity increased with the pH value between 6 and 7 and remained in a comparable level in Hepes buffer between pH 7 and 8. TTLL4 activity was reduced when pH was higher than 8.5 (Fig. 2A). Next, we examined how ion strength may affect the activity of the recombinant TTLL4. TTLL4 activity was reduced with increased concentration of NaCl. With the presence of 50 or 100 mM NaCl, the enzyme activity was inhibited by about 25% and 50%, respectively (Fig. 2B). TTLL4 activity was increased when KCl concentration was lower than 100 mM but was inhibited by KCl at higher concentrations (Fig. 2C). These properties are similar to those of recombinant TTLL7 (17Mukai M. Ikegami K. Sugiura Y. Takeshita K. Nakagawa A. Setou M. Recombinant mammalian tubulin polyglutamylase TTLL7 performs both initiation and elongation of polyglutamylation on β-tubulin through a random sequential pathway.Biochemistry. 2009; 48: 1084-1093Crossref PubMed Scopus (32) Google Scholar). The activity of TTLLs may depend on the polymerization status of tubulin (2van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. A targeted multienzyme mechanism for selective microtubule polyglutamylation.Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 17Mukai M. Ikegami K. Sugiura Y. Takeshita K. Nakagawa A. Setou M. Recombinant mammalian tubulin polyglutamylase TTLL7 performs both initiation and elongation of polyglutamylation on β-tubulin through a random sequential pathway.Biochemistry. 2009; 48: 1084-1093Crossref PubMed Scopus (32) Google Scholar). For instance, the recombinant TTLL7 can glutamylate MTs more efficiently than free tubulins (17Mukai M. Ikegami K. Sugiura Y. Takeshita K. Nakagawa A. Setou M. Recombinant mammalian tubulin polyglutamylase TTLL7 performs both initiation and elongation of polyglutamylation on β-tubulin through a random sequential pathway.Biochemistry. 2009; 48: 1084-1093Crossref PubMed Scopus (32) Google Scholar, 34Garnham C.P. Vemu A. Wilson-Kubalek E.M. Yu I. Szyk A. Lander G.C. et al.Multivalent microtubule recognition by tubulin tyrosine ligase-like family glutamylases.Cell. 2015; 161: 1112-1123Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). We wondered whether tubulin polymerization could also affect the activity of TTLL4. NB tubulins or their taxol-stabilized MTs were incubated with TTLL4 separately. We found that the free tubulin and MTs were glutamylated at similar levels after 1 h incubation (Fig. 2, D and E). Therefore, the recombinant TTLL4 can equally process free tubulins of NB and their polymerized MTs. GT335 antibody was generated using a peptide corresponding to the C terminus of α-tubulin with two glutamate residues in the side chain (37Wolff A. de Néchaud B. Chillet D. Mazarguil H. Desbruyères E. Audebert S. et al.Distribution of glutamylated alpha and beta-tubulin in mouse tissues using a specific monoclonal antibody, GT335.Eur. J. Cell Biol. 1992; 59: 425-432PubMed Google Scholar). It also recognizes modified β-tubulin and nontubulin substrates (2van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. A targeted multienzyme mechanism for selective microtubule polyglutamylation.Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 11Berezniuk I. Vu H.T. Lyons P.J. Sironi
Abstract Micropeptides encoded by short open reading frames (sORFs) within long noncoding RNAs (lncRNAs) are beginning to be discovered and characterized as regulators of biological and pathological processes. Here, we find that lncRNA Dleu2 encodes a 17-amino-acid micropeptide, which we name Dleu2-17aa, that is abundantly expressed in T cells. Dleu2-17aa promotes inducible regulatory T (iTreg) cell generation by interacting with SMAD Family Member 3 (Smad3) and enhancing its binding to the Foxp3 conserved non-coding DNA sequence 1 (CNS1) region. Importantly, the genetic deletion of Dleu2-17aa in mice by start codon mutation impairs iTreg generation and worsens experimental autoimmune encephalomyelitis (EAE). Conversely, the exogenous supplementation of Dleu2-17aa relieves EAE. Our findings demonstrate an indispensable role of Dleu2-17aa in maintaining immune homeostasis and suggest therapeutic applications for this peptide in treating autoimmune diseases.
Regulatory T (Treg) cells constitute a dynamic population that is critical in autoimmunity. Treg cell therapies for autoimmune diseases are mainly focused on enhancing their suppressive activities. However, recent studies demonstrated that certain inflammatory conditions induce Treg cell instability with diminished FoxP3 expression and convert them into pathogenic effector cells. Therefore, the identification of novel targets crucial to both Treg cell function and plasticity is of vital importance to the development of therapeutic approaches in autoimmunity. In this study, we found that conditional Pp6 knockout (cKO) in Treg cells led to spontaneous autoinflammation, immune cell activation, and diminished levels of FoxP3 in CD4+ T cells in mice. Loss of Pp6 in Treg cells exacerbated two classical mouse models of Treg-related autoinflammation. Mechanistically, Pp6 deficiency increased CpG motif methylation of the FoxP3 locus by dephosphorylating Dnmt1 and enhancing Akt phosphorylation at Ser473/Thr308, leading to impaired FoxP3 expression in Treg cells. In summary, our study proposes Pp6 as a critical positive regulator of FoxP3 that acts by decreasing DNA methylation of the FoxP3 gene enhancer and inhibiting Akt signaling, thus maintaining Treg cell stability and preventing autoimmune diseases.
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