Posttranslational Modifications Mediate the Structural Diversity of Tauopathy Strains.

Tau aggregation into insoluble filaments is the defining pathological hallmark of tauopathies. However, it is not known what controls the formation and templated seeding of strain-specific structures associated with individual tauopathies. Here, we use cryo-electron microscopy (cryo-EM) to determine the structures of tau filaments from corticobasal degeneration (CBD) human brain tissue. Cryo-EM and mass spectrometry of tau filaments from CBD reveal that this conformer is heavily decorated with posttranslational modifications (PTMs), enabling us to map PTMs directly onto the structures. By comparing the structures and PTMs of tau filaments from CBD and Alzheimer's disease, it is found that ubiquitination of tau can mediate inter-protofilament interfaces. We propose a structure-based model in which cross-talk between PTMs influences tau filament structure, contributing to the structural diversity of tauopathy strains. Our approach establishes a framework for further elucidating the relationship between the structures of polymorphic fibrils, including their PTMs, and neurodegenerative disease.

Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization.

Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.

Middle-Down Proteomics Reveals Dense Sites of Methylation and Phosphorylation in Arginine-Rich RNA-Binding Proteins.

Post-translational modifications (PTMs) within arginine (Arg)-rich RNA-binding proteins, such as phosphorylation and methylation, regulate multiple steps in RNA metabolism. However, the identification of PTMs within Arg-rich domains with complete trypsin digestion is extremely challenging due to the high density of Arg residues within these proteins. Here, we report a middle-down proteomic approach coupled with electron-transfer dissociation (ETD) mass spectrometry to map previously unknown sites of phosphorylation and methylation within the Arg-rich domains of U1-70K and structurally similar RNA-binding proteins from nuclear extracts of human embryonic kidney (HEK)-293T cells. Notably, the Arg-rich domains in RNA-binding proteins are densely modified by methylation and phosphorylation compared with the remainder of the proteome, with methylation and phosphorylation favoring RSRS motifs. Although they favor a common motif, analysis of combinatorial PTMs within RSRS motifs indicates that phosphorylation and methylation do not often co-occur, suggesting that they may functionally oppose one another. Furthermore, we show that phosphorylation may modify interactions between Arg-rich proteins, as serine-arginine splicing factor 2 (SRSF2) has a stronger association with U1-70K and LUC7L3 upon dephosphorylation. Collectively, these findings suggest that the level of PTMs within Arg-rich domains may be among the highest in the proteome and a possible unexplored regulator of RNA-binding protein interactions.

RNA-binding proteins with basic-acidic dipeptide (BAD) domains self-assemble and aggregate in Alzheimer's disease.

The U1 small nuclear ribonucleoprotein 70 kDa (U1-70K) and other RNA-binding proteins (RBPs) are mislocalized to cytoplasmic neurofibrillary Tau aggregates in Alzheimer's disease (AD), yet the co-aggregation mechanisms are incompletely understood. U1-70K harbors two disordered low-complexity domains (LC1 and LC2) that are necessary for aggregation in AD brain extracts. The LC1 domain contains highly repetitive basic (Arg/Lys) and acidic (Asp/Glu) residues, referred to as a basic-acidic dipeptide (BAD) domain. We report here that this domain shares many of the properties of the Gln/Asn-rich LC domains in RBPs that also aggregate in neurodegenerative disease. These properties included self-assembly into oligomers and localization to nuclear granules. Co-immunoprecipitations of recombinant U1-70K and deletions lacking the LC domain(s) followed by quantitative proteomic analyses were used to resolve functional classes of U1-70K-interacting proteins that depend on the BAD domain for their interaction. Within this interaction network, we identified a class of RBPs with BAD domains nearly identical to that found in U1-70K. Two members of this class, LUC7L3 and RBM25, required their respective BAD domains for reciprocal interactions with U1-70K and nuclear granule localization. Strikingly, a significant proportion of RBPs with BAD domains had elevated insolubility in the AD brain proteome. Furthermore, we show that the BAD domain of U1-70K can interact with Tau from AD brains but not from other tauopathies. These findings highlight a mechanistic role for BAD domains in stabilizing RBP interactions and in potentially mediating co-aggregation with the pathological AD-specific Tau isoforms.

Cos2/Kif7 and Osm-3/Kif17 regulate onset of outer segment development in zebrafish photoreceptors through distinct mechanisms.

Zebrafish morphants of osm-3/kif17, a kinesin-2 family member and intraflagellar transport motor, have photoreceptor outer segments that are dramatically reduced in number and size. However, two genetic mutant lines, osm-3/kif17sa0119 and osm-3/kif17sa18340, reportedly lack any observable morphological outer segment defects. In this work, we use TALENs to generate an independent allele, osm-3/kif17mw405, and show that both osm-3/kif17sa0119 and osm-3/kif17mw405 have an outer segment developmental delay in both size and density that is fully recovered by 6 days post-fertilization. Additionally, we use CRISPRs to generate cos2/kif7mw406, a mutation in the kinesin-4 family member cos2/kif7 that has been implicated in controlling ciliary architecture and Hedgehog signaling to test whether it may be functioning redundantly with osm-3/kif17. We show that cos2/kif7mw406 has an outer segment developmental delay similar to the osm-3/kif17 mutants. Using a three-dimensional mathematical model of outer segments, we show that while cos2/kif7mw406 and osm-3/kif17mw405 outer segments are smaller throughout the first 6 days of development, the volumetric rates of outer segment morphogenesis are not different among wild-type, cos2/kif7mw406, and osm-3/kif17mw405 after 60hpf. Instead, our model suggests that cos2/kif7mw406 and osm-3/kif17mw405 impact outer segment morphogenesis through upstream events that that are different for each motor. In the case of cos2/kif7mw406 mutants, we show that early defects in Hedgehog signaling lead to a general, non-photoreceptor-specific delay of retinal neurogenesis, which in turn causes the secondary phenotype of delayed outer segment morphogenesis. In contrast, the osm-3/kif17mw405 outer segment morphogenesis delays are linked specifically to initial disc morphogenesis of photoreceptors rather than an upstream event. Further, we show that osm-3/kif17 mutant mice also exhibit a similarly delayed outer segment development, suggesting a role for osm-3/kif17 in early outer segment development that is conserved across species. In conclusion, we show that both osm-3/kif17 and cos2/kif7 have comparable outer segment developmental delays, although through independent mechanisms.

Kif17 phosphorylation regulates photoreceptor outer segment turnover.

BACKGROUND: KIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown. RESULTS: Using transgenic expression in zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization along the cone outer segment. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding. CONCLUSIONS: Taken together, our data support a model in which phosphorylation of KIF17 promotes its photoreceptor outer segment localization and disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.

Targeted Quantification of Detergent-Insoluble RNA-Binding Proteins in Human Brain Reveals Stage and Disease Specific Co-aggregation in Alzheimer's Disease.

Core spliceosome and related RNA-binding proteins aggregate in Alzheimer's disease (AD) brain even in early asymptomatic stages (AsymAD) of disease. To assess the specificity of RNA-binding protein aggregation in AD, we developed a targeted mass spectrometry approach to quantify broad classes of RNA-binding proteins with other pathological proteins including tau and amyloid beta (Aß) in detergent insoluble fractions from control, AsymAD, AD and Parkinson's disease (PD) brain. Relative levels of specific insoluble RNA-binding proteins across different disease groups correlated with accumulation of Aß and tau aggregates. RNA-binding proteins, including splicing factors with homology to the basic-acidic dipeptide repeats of U1-70K, preferentially aggregated in AsymAD and AD. In contrast, PD brain aggregates were relatively depleted of many RNA-binding proteins compared to AsymAD and AD groups. Correlation network analyses resolved 29 distinct modules of co-aggregating proteins including modules linked to spliceosome assembly, nuclear speckles and RNA splicing. Modules related to spliceosome assembly and nuclear speckles showed stage-specific enrichment of insoluble RBPs from AsymAD and AD brains, whereas the RNA splicing module was reduced specifically in PD. Collectively, this work identifies classes of RNA-binding proteins that distinctly co-aggregate in detergent-insoluble fractions across the specific neurodegenerative diseases we examined.

Global quantitative analysis of the human brain proteome and phosphoproteome in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by an early, asymptomatic phase (AsymAD) in which individuals exhibit amyloid-beta (Aß) plaque accumulation in the absence of clinically detectable cognitive decline. Here we report an unbiased multiplex quantitative proteomic and phosphoproteomic analysis using tandem mass tag (TMT) isobaric labeling of human post-mortem cortex (n = 27) across pathology-free controls, AsymAD and symptomatic AD individuals. With off-line high-pH fractionation and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on an Orbitrap Lumos mass spectrometer, we identified 11,378 protein groups across three TMT 11-plex batches. Immobilized metal affinity chromatography (IMAC) was used to enrich for phosphopeptides from the same TMT-labeled cases and 51,736 phosphopeptides were identified. Of these, 48,992 were quantified by TMT reporter ions representing 33,652 unique phosphosites. Two reference standards in each TMT 11-plex were included to assess intra- and inter-batch variance at the protein and peptide level. This comprehensive human brain proteome and phosphoproteome dataset will serve as a valuable resource for the identification of biochemical, cellular and signaling pathways altered during AD progression.