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Pathological TDP‐43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD‐TDP); however, how TDP‐43 aggregation and function are regulated remain poorly understood. Here, we show that O‐GlcNAc transferase OGT‐mediated O‐GlcNAcylation of TDP‐43 suppresses ALS‐associated proteinopathies and promotes TDP‐43's splicing function. Biochemical and cell‐based assays indicate that OGT's catalytic activity suppresses TDP‐43 aggregation and hyperphosphorylation, whereas abolishment of TDP‐43 O‐GlcNAcylation impairs its RNA splicing activity. We further show that TDP‐43 mutations in the O‐GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP‐43 overexpression in Drosophila motor neurons. We finally demonstrate that O‐GlcNAcylation of TDP‐43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. Our findings suggest that O‐GlcNAcylation might be a target for the treatment of TDP‐43‐linked pathogenesis.

Ubiquitin-specific protease 19 (USP19) is a member of the deubiquitinating (DUB) enzymes that catalyze removing the ubiquitin signals from target proteins. Our previous research has demonstrated that USP19 up-regulates the protein level and aggregation of polyQ-expanded huntingtin through the involvement of heat shock protein 90 (HSP90). Here, we present solution structures of the CS1, CS2 and UbL domains of USP19 and structural insights into their domain interactions. We found that the tandem CS domains fold back to interact with the C-terminal USP domain (USPD) intra-molecularly that leads to inhibition of the catalytic core of USP19, especially CS1 interacts with the embedded UbL domain and CS2 does with the CH2 catalytic core. Moreover, CS2 specifically interacts with the NBD domain of HSP90, which can activate the DUB enzyme. A mechanism of auto-inhibition of USP19 and activation by HSP90 is proposed, on which USP19 modulates the protein level of polyQ-expanded huntingtin in cells. This study provides structural and mechanistic insights into the modulation of protein level and aggregation by USP19 with the assistance of HSP90.

TDP-43 is a primary pathological hallmark protein of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, which may exist in the form of amyloid inclusions in the cells of patients. In addition to serving as a biomarker for these diseases, TDP-43 can also directly trigger neurodegeneration. We previously determined the amyloidogenic core region of TDP-43 (residues 311-360) and showed by solution NMR that this region includes two α-helices [(321-330) and (335-343)] in solution. We suggested that the helix-to-sheet structural transformation initiates TDP-43 aggregation. In the present study, X-ray diffraction shows that TDP-43 (311-360) aggregates adopt a cross-β structure. Thioredoxin (Trx)-fused TDP-43 (311-360) can undergo liquid-liquid phase separation (LLPS) before fibrillation, suggesting that phase separation is an intermediate step before amyloid formation. Solid-state NMR (SSNMR), carried out to elucidate the structural changes of TDP-43 (311-360) at the atomic level, indicates five β-strands of the amyloids formed, with the major two β-strands contributed by the first helical region in the solution structure. The NMR evidence is also in support of the fibril having a parallel in-register conformation, implying a mechanism in which the helix-helix interactions in LLPS are converted into β-strand parallel lateral association upon fibrillation. Our studies have assigned many key interresidue interactions that contribute to the stability of the fibril, including F316 with I318 and Q327 and W334 with A325, A326, A329, and S332. SSNMR with 1H detection reveals a unique close interaction between the indole Ne1-He1 of W334 and the side-chain carbonyl of Q343. This interaction could be a very important factor in initiating TDP-43 (311-360) folding/misfolding in LLPS.

The components of ubiquitin (Ub)-proteasome system, such as Ub, Ub adaptors, or proteasome subunits, are commonly accumulated with the aggregated proteins in inclusions, but how protein aggregates sequester Ub-related proteins remains elusive. Using N-terminal huntingtin (Htt-N552) and ataxin (Atx)-3 as model proteins, we investigated the molecular mechanism underlying sequestration of Ub adaptors by polyQ-expanded proteins. We found that polyQ-expanded Htt-N552 and Atx-3 sequester endogenous Ub adaptors, human RAD23 homolog B (hHR23B) and ubiquilin (UBQLN)-2, into inclusions. This sequestration effect is dependent on the UBA domains of Ub adaptors and the conjugated Ub of the aggregated proteins. Moreover, polyQ-expanded Htt-N552 and Atx-3 reduce the protein level of xeroderma pigmentosum group C (XPC) by sequestration of hHR23B, suggesting that this process may cut down the available quantity of hHR23B and thus affect its normal function in stabilizing XPC. Our findings demonstrate that polyQ-expanded proteins sequester Ub adaptors or other Ub-related proteins into aggregates or inclusions through ubiquitination of the pathogenic proteins. This study may also provide a common mechanism for the formation of Ub-positive inclusions in cells.-Yang, H., Yue, H.-W., He, W.-T., Hong, J.-Y., Jiang, L.-L., Hu, H.-Y. PolyQ-expanded huntingtin and ataxin-3 sequester ubiquitin adaptors hHR23B and UBQLN2 into aggregates via conjugated ubiquitin.

Ataxin-7 (Atx7) is a disease-related protein associated with the pathogenesis of spinocerebellar ataxia 7, while its polyglutamine (polyQ) tract in N-terminus is the causative source of aggregation and proteinopathy. We investigated the structure, dynamics and aggregation properties of the N-terminal 62-residue fragment of Atx7 (Atx7-N) by biochemical and biophysical approaches. The results showed that the normal Atx7-N with a tract of 10 glutamines (10Q) overall adopts a flexible and disordered structure, but it may contain a short or small population of helical structure in solution. PolyQ expansion increases the α-helical propensity of the polyQ tract and consequently enhances its transformation into β-sheet structures during amyloid aggregation. An alanine-rich region (ARR) just ahead of the polyQ tract forms a local and relatively stable α-helix. The ARR α-helix can initiate and stabilize helical formation of the following polyQ tract, but it may suppress aggregation of the polyQ-expanded Atx7-N both in vitro and in cell. Thus, the preceding ARR segment in Atx7-N may influence the dynamic structure and aggregation property of the polyQ tract and even determine the threshold of the pathogenic polyQ lengths. This study may gain structural and dynamic insights into amyloid aggregation of Atx7 and help us further understand the Atx7 proteinopathy based on polyQ expansion.

TDP-43 is a nuclear factor that functions in promoting pre-mRNA splicing. Deletion of the N-terminal domain (NTD) and nuclear localization signal (NLS) (i.e., TDP-35) results in mislocalization to cytoplasm and formation of inclusions. However, how the NTD functions in TDP-43 activity and proteinopathy remains largely unknown. Here, we studied the structure and function of the NTD in inclusion formation and pre-mRNA splicing of TDP-43 by using biochemical and biophysical approaches. We found that TDP-43 NTD forms a homodimer in solution in a concentration-dependent manner, and formation of intermolecular disulfide results in further tetramerization. Based on the NMR structure of TDP-43 NTD, the dimerization interface centered on Leu71 and Val72 around the β7-strand was defined by mutagenesis and size-exclusion chromatography. Cell experiments revealed that the N-terminal dimerization plays roles in protecting TDP-43 against formation of cytoplasmic inclusions and enhancing pre-mRNA splicing activity of TDP-43 in nucleus. This study may provide mechanistic insights into the physiological function of TDP-43 and its related proteinopathies.

TDP-43 is a DNA/RNA binding protein associated with TDP-43 proteinopathies. Many mutations have been identified in the flexible C-terminal region, which is implicated in the disease pathology. We investigated four point mutations in the amyloidogenic core region (residues 311–360) of TDP-43 by biochemical and spectroscopic methods. We found that the G335D mutation enhances the aggregation and inclusion formation of TDP-43 and this mutant in TDP-35 (the C-terminal fragment of 35 kDa) exaggerates the antagonist effect on RNA processing by endogenous TDP-43; whereas Q343R gives an opposite effect. As a comparison, M337V and Q331K have very little impact on the aggregation and inclusion formation of TDP-43 or TDP-35. NMR structural analysis showed that the G335D mutant in the core region forms a loop linker between the two α-helices and promotes α-to-β transition, but Q343R loses the second helix and consequently the structural transformation. Thus, the propensity of structural transformation in the amyloidogenic core of TDP-43 determines its aggregation and inclusion formation. This study may provide a molecular mechanism of the TDP-43 proteinopathies caused by genetic mutations.

TDP-43 (TAR DNA binding protein of 43kDa) and its C-terminal fragments are thought to be linked to the pathologies of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Here, we demonstrate that the aggregates or inclusions formed by its 35-kDa fragment (namely TDP-35) sequester full-length TDP-43 into cytoplasmic inclusions; and this sequestration is mediated by binding with RNA that is enriched in the cytoplasmic inclusions. RNA recognition motif 1 (RRM1) of TDP-43/TDP-35 plays a dominant role in nucleic-acid binding; mutation in this moiety abrogates formation of the TDP-35 inclusions and its RNA-assisted association with TDP-43. Thus, TDP-35 is able to sequester TDP-43 from nuclear localization into cytoplasmic inclusions, and RNA binding plays an essential role in this process.

Human ataxin 7 (Atx7) is a component of the deubiquitination module (DUBm) in the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex for transcriptional regulation, and expansion of its polyglutamine (polyQ) tract leads to spinocerebellar ataxia type 7. However, how polyQ expansion of Atx7 affects DUBm function remains elusive. We investigated the effects of polyQ-expanded Atx7 on ubiquitin-specific protease (USP22), an interacting partner of Atx7 functioning in deubiquitination of histone H2B. The results showed that the inclusions or aggregates formed by polyQ-expanded Atx7 specifically sequester USP22 through their interactions mediated by the N-terminal zinc finger domain of Atx7. The mutation of the zinc finger domain in Atx7 that disrupts its interaction with USP22 dramatically abolishes sequestration of USP22. Moreover, polyQ expansion of Atx7 decreases the deubiquitinating activity of USP22 and, consequently, increases the level of monoubiquitinated H2B. Therefore, we propose that polyQ-expanded Atx7 forms insoluble aggregates that sequester USP22 into a catalytically inactive state, and then the impaired DUBm loses the function to deubiquitinate monoubiquitinated histone H2B or H2A. This may result in dysfunction of the SAGA complex and transcriptional dysregulation in spinocerebellar ataxia type 7 disease.

TDP-43 (TAR DNA-binding protein of 43 kDa) is a major deposited protein in amyotrophic lateral sclerosis and frontotemporal dementia with ubiquitin. A great number of genetic mutations identified in the flexible C-terminal region are associated with disease pathologies. We investigated the molecular determinants of TDP-43 aggregation and its underlying mechanisms. We identified a hydrophobic patch (residues 318–343) as the amyloidogenic core essential for TDP-43 aggregation. Biophysical studies demonstrated that the homologous peptide formed a helix-turn-helix structure in solution, whereas it underwent structural transformation from an α-helix to a β-sheet during aggregation. Mutation or deletion of this core region significantly reduced the aggregation and cytoplasmic inclusions of full-length TDP-43 (or TDP-35 fragment) in cells. Thus, structural transformation of the amyloidogenic core initiates the aggregation and cytoplasmic inclusion formation of TDP-43. This particular core region provides a potential therapeutic target to design small-molecule compounds for mitigating TDP-43 proteinopathies.

TAR DNA binding protein of 43 kDa (TDP-43) is a nuclear factor functioning in RNA processing. It is also a major deposited protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin (FTLD-U). To understand the mechanism underlying the inclusion body formation and possible functional alteration, we studied some TDP-43 fragments and their effects on RNA processing in cell models. The results show that the 35-kDa fragment of TDP-43 (namely TDP-35, residues 90-414), but not TDP-25A (184-414), is capable of forming cytoplasmic inclusion bodies and altering pre-mRNA splicing. The inclusions formed by TDP-35 can also recruit full-length TDP-43 to cytoplasmic deposition from functionally nuclear localization. The in vitro studies demonstrate that TDP-35, rather than TDP-43 and TDP-25A, is prone to aggregation, and it further serves as a seed to facilitate aggregation of full-length TDP-43. This suggests that fragmentation of TDP-43 leads to cellular redistribution, inclusion body formation, and altered RNA processing, which are implicated in the molecular pathogenesis of ALS and FTLD.