Polyamination with spermidine enhances pathogenic tau conformations while reducing filamentous aggregate formation in vitro

Tau is subject to a broad range of post-translational modifications (PTMs) that regulate its biological activity in health and disease, including microtubule (MT) dynamics, aggregation, and adoption of pathogenic conformations. The most studied PTMs of tau are phosphorylation and acetylation; however, the salience of other PTMs is not fully explored. Tissue transglutaminase (TG) is an enzyme whose activity is elevated in Alzheimer’s disease (AD). TG action on tau may lead to intramolecular and intermolecular cross-linking along with the incorporation of cationic polyamines [e.g., spermidine (SPD)] onto glutamine residues (Q). Even though SPD levels are significantly elevated in AD, the effects of SPD polyamination on tau biology have yet to be examined. In this work, we describe a method to produce recombinant SPD-modified tau where SPD modifications are mainly localized to Q residues within the N-terminus.  MT binding and polymerization assays showed that SPD modification does not..., Preparation of recombinant unmodified and SPD-modified tau proteins
Recombinant hT40 and hT39 tau proteins were prepared from a 4L bacterial culture as described previously (Combs et al., 2017), with the exception that BL21 bacteria (NEB, #C2527H) were used. The concentration of recombinant tau protein was determined using the BCA method (Thermo, # A53225). Next, polyamination reactions were performed in vitro by adapting the protocol described by Song and coworkers (Song et al., 2013). Briefly, 8 mM of SPD (Sigma, # S2626-1G) and 0.2 µM of TG enzyme (Sigma, # TS398) were added to 0.93 mg/ml of tau in 50 µl of reaction buffer (50mM Tris HCl, pH 8, 10 mM calcium chloride and 5 mM DTT) followed by a 1-hour incubation at 37 °C. Unmodified tau proteins were subjected to the same reaction conditions, but TG was excluded. Then, the TG enzyme was inactivated by heating at 70 °C for 2 min. The TG-catalyzed reaction produces SPD-modified monomeric proteins as well as intra- and inter-molecular c..., , # Polyamination with spermidine enhances pathological tau conformations while reducing filamentous aggregate formation in vitro

[https://doi.org/10.5061/dryad.59zw3r2jp](https://doi.org/10.5061/dryad.59zw3r2jp)

## Description of the data and file structure

These data are mass spectrometry RAW data files and full data sets from MetaMorpheus analysis output derived from analyzing recombinant human tau proteins (hT40 - 4R tau and hT39 - 3R tau) in E. coli that were either unmodified or modified by transglutaminase-mediated polyamination (with spermidine) *in vitro*. The recombinant proteins were purified using a series of chromatography approaches prior to and after polyamination reactions. The samples were digested with trypsin and rLysC and analyzed by mass spectrometry for spermidine modifications on Q residues. 

### Files and variables

#### File: SPD\_Modified\_3R\_Tau.raw

**Description:** RAW data files for SPD-modified recombinant 3R tau protein

#### File: 4R\_MetaMorph\_Outpu...,