Biology subjects

Kaniyappan, S.

Publications and source records attributed to Kaniyappan, S..

4 recordsLinked to original sources

bioRxiv2022

Biomolecular Tau condensation is linked to Tau accumulation at the nuclear envelope

Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, this synergy enhances bundling and spatially arranges microtubules. We further show that different Tau condensates efficiently induce pathological Tau in cells, including small accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations, and a condensate-like density for nuclear envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations.

molecular biology↗

bioRxiv2020

Combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications

Abnormal changes in the neuronal microtubule-associated protein Tau, such as hyperphosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer disease. Hyperphosphorylation is thought to take place before aggregation, and therefore it is often assumed that phosphorylation predisposes Tau towards aggregation. However, the nature and extent of phosphorylation has remained ill-defined. Tau protein contains up to 85 potential phosphorylation sites (80 Ser/Thr, and 5 Tyr P-sites), many of which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, limitations in methods (e.g. in mass spectrometry of phosphorylated peptides, or antibodies against phospho-epitopes) have led to conflicting results regarding the overall degree of phosphorylation of Tau in cells. Here we present results from a new approach, that is based on native mass spectrometry analysis of intact Tau expressed in a eukaryotic cell system (Sf9) which reveals Tau in different phosphorylation states. The extent of phosphorylation is remarkably heterogeneous with up to [~]20 phosphates (Pi) per molecule and distributed over 51 sites (including all P-sites published so far and additional 18 P-sites). The medium phosphorylated fraction Pm showed overall occupancies centered at 8 Pi ({+/-} 5 Pi) with a bell-shaped distribution, the highly phosphorylated fraction Ph had 14 Pi ({+/-} 6 Pi). The distribution of sites was remarkably asymmetric (with 71% of all P-sites located in the C-terminal half of Tau). All phosphorylation sites were on Ser or Thr residues, but none on Tyr. Other known posttranslational modifications of Tau were near or below our detection limit (e.g. acetylation, ubiquitination). None of the Tau fractions self-assemble readily, arguing that Tau aggregation is not promoted by phosphorylation per se but requires additional factors.

neuroscience↗

bioRxiv2020

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau fibers

Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism proposed for prion pathogenesis. In cell cultures, the process is usually monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (TauRD, with pro-aggregant mutation, e.g., {Delta}K280 or P301L, [~]13.5 kDa) fused to GFP-based FRET pairs (YFP or CFP, [~]28 kD). Since the diameter of the reporter GFP ([~]3 nm) is [~]6.5 times larger than the {beta}-strand distance (0.47nm), this points to a potential steric clash. Hence, we investigated the influence of GFP tagged (N- or C-terminally) TauRD and TauFL (full-length Tau) on their aggregation behavior in vitro. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRD{Delta}K-GFP was severely inhibited, even in the presence of nucleation enhancers (heparin and/or pre-formed PHFs from TauRD{Delta}K). Some rare fiber-like particles had a very different subunit packing from proper PHFs, as judged by STEM. The mass per length (MPL) values of TauRD{Delta}K fibrils are equivalent to 4.45 molecules/nm, close to the expected value for a paired-helical fiber with 2 protofilaments and cross-{beta} structure. By contrast, the elongated particles formed by TauRD{Delta}K-GFP have MPL values around [~]2, less than half of the values expected for PHFs, indicating that the subunit packing is distinct. Thus, both kinetic and structural observations are incompatible with a model whereby external Tau can form a template for PHF assembly of Tau-GFP in recipient cells. As a consequence, the observed local increase of FRET in recipient cells must be caused by other signalling processes.

neuroscience↗

bioRxiv2019

The plasma Factor XIII heterotetrameric complex structure: unexpected unequal pairing within a symmetric complex

Factor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with 2 catalytic A subunits and 2 carrier-protective B subunits in a heterotetramer (FXIII-A2B2). FXIII-A2 and -B2 subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B-subunits, activated FXIII (FXIIIa) covalently cross-links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A2B2 complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A2B2 complex. We also used molecular dynamic simulations in combination with isothermal titration calorimetry to characterize FXIII-A2B2 assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A2B2 mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly and/or activity to reduce bleeding and thrombotic complications.

molecular biology↗