Biology subjects

Mullapudi, V.

Publications and source records attributed to Mullapudi, V..

7 recordsLinked to original sources

bioRxiv2023

DnaJC7 specifically regulates tau seeding

Neurodegenerative tauopathies are caused by accumulation of toxic tau protein assemblies. This appears to involve template-based seeding events, whereby tau monomer changes conformation and is recruited to a growing aggregate. Several large families of chaperone proteins, including Hsp70s and J domain proteins (JDPs) cooperate to regulate the folding of intracellular proteins such as tau, but the factors that coordinate this activity are not well known. The JDP DnaJC7 binds tau and reduces its intracellular aggregation. However, it is unknown whether this is specific to DnaJC7 or if other JDPs might be similarly involved. We used proteomics within a cell model to determine that DnaJC7 co-purified with insoluble tau and colocalized with intracellular aggregates. We individually knocked out every possible JDP and tested the effect on intracellular aggregation and seeding. DnaJC7 knockout decreased aggregate clearance and increased intracellular tau seeding. This depended on the ability of the J domain (JD) of DnaJC7 to bind to Hsp70, as JD mutations that block binding to Hsp70 abrogated the protective activity. Disease-associated mutations in the JD and substrate binding site of DnaJC7 also abrogated its protective activity. DnaJC7 thus specifically regulates tau aggregation in cooperation with Hsp70.

molecular biology↗

bioRxiv2023

Disease-associated patterns of acetylation stabilize tau fibril formation

Assembly of the microtubule-associated protein into tauopathy fibril conformations dictates the pathology of a diversity of diseases. Recent cryogenic Electron Microscopy (cryo-EM) structures have uncovered distinct fibril conformations in different tauopathies but it remains unknown how these structures fold from a single protein sequence. It has been proposed that post-translational modifications may drive tau assembly but no direct mechanism for how modifications drive assembly has emerged. Leveraging established aggregation-regulating tau fragments that are normally inert, we tested the effect of chemical modification of lysines with acetyl groups on tau fragment conversion into amyloid aggregates. We identify specific patterns of acetylation that flank amyloidogenic motifs on the tau fragments that drive rapid fibril assembly. To understand how this pattern of acetylation may drive assembly, we determined a 3.9 [A] cryo-EM structure of an amyloid fibril assembled from an acetylated tau fragment. The structure uncovers how lysine acetylation patterns mediate gain-of-function interactions to promote amyloid assembly. Comparison of the structure to an ex vivo tau fibril conformation from Picks Disease reveals regions of high structural similarity. Finally, we show that our lysine- acetylated sequences exhibit fibril assembly activity in cell-based tau aggregation assays. Our data uncover the dual role of lysine residues in limiting aggregation while their acetylation leads to stabilizing pro-aggregation interactions. Design of tau sequence with specific acetylation patterns may lead to controllable tau aggregation to direct folding of tau into distinct folds.

biophysics↗

bioRxiv2022

FTD-tau S320F mutation stabilizes local structure and allosterically promotes amyloid motif-dependent aggregation

Amyloid deposition of the microtubule-associated protein tau is a unifying theme in a multitude of neurodegenerative diseases. Disease-associated missense mutations in tau are associated with frontotemporal dementia (FTD) and enhance tau aggregation propensity. However, the molecular mechanism of how mutations in tau promote tau assembly into amyloids remains obscure. There is a need to understand how tau folds into pathogenic conformations to cause disease. Here we describe the structural mechanism for how an FTD-tau S320F mutation drives spontaneous aggregation. We use recombinant protein and synthetic peptide systems, computational modeling, cross-linking mass spectrometry, and cell models to investigate the mechanism of spontaneous aggregation of the S320F FTD-tau mutant. We discover that the S320F mutation drives the stabilization of a local hydrophobic cluster which allosterically exposes the 306VQIVYK311 amyloid motif. We identify a suppressor mutation that reverses the S320F aggregation phenotype through the reduction of S320F-based hydrophobic clustering in vitro and in cells. Finally, we use structure-based computational design to engineer rapidly aggregating tau sequences by optimizing nonpolar clusters in proximity to the S320 site revealing a new principle that governs the regulation of tau aggregation. We uncover a mechanism for regulating aggregation that balances transient nonpolar contacts within local protective structures or in longer-range interactions that sequester amyloid motifs. The introduction of a pathogenic mutation redistributes these transient interactions to drive spontaneous aggregation. We anticipate deeper knowledge of this process will permit control of tau aggregation into discrete structural polymorphs to aid design of reagents that can detect disease-specific tau conformations.

biophysics↗

bioRxiv2022

Network of hotspot interactions cluster tau amyloid folds

Cryogenic electron microscopy has revealed unprecedented molecular insight into the conformation of {beta}-sheet-rich protein amyloids linked to neurodegenerative diseases. It remains unknown how a protein can adopt a diversity of folds and form multiple distinct fibrillar structures. Here we develop an in silico alanine scan method to estimate the relative energetic contribution of each amino acid in an amyloid assembly. We apply our method to twenty-seven ex vivo and in vitro fibril structural polymorphs of the microtubule-associated protein tau. We uncover networks of energetically important interactions involving amyloid-forming motifs that stabilize the different fibril folds. We test our predictions in cellular and in vitro aggregation assays. Using a machine learning approach, we classify the structures based on residue energetics to identify distinguishing and unifying features. Our energetic profiling suggests that minimal sequence elements that control the stability of tau fibrils, allowing future design of protein sequences that fold into unique structures.

neuroscience↗

bioRxiv2022

Development of a new DHFR-based destabilizing domain with enhanced basal turnover and applicability in mammalian systems

Destabilizing domains (DDs) are an attractive strategy allowing for positive post-transcriptional small molecule-regulatable control of a fusion proteins abundance. Yet in many instances, the currently available DDs suffer from higher-than-desirable basal levels of the fusion protein. Accordingly, we redesigned the E. coli dihydrofolate reductase (ecDHFR) DD by introducing a library of ~1200 random ecDHFR mutants fused to YFP into CHO cells. Following successive rounds of FACS sorting, we identified six new ecDHFR DD clones with significantly enhanced proteasomal turnover in the absence of a stabilizing ligand, trimethoprim (TMP). One of these clones, designated as C12, contained four unique missense mutations (W74R/T113S/E120D/Q146L) and demonstrated a significant 2.9-fold reduction in basal levels compared to the conventional ecDHFR DD YFP. This domain was similarly responsive to TMP with respect to dose-response and maximal stabilization, indicating an overall enhanced dynamic range. Interestingly, both computational and wet-lab experiments identified the W74R and T113S mutations of C12 as the main contributors towards its basal destabilization. Yet, the combination of all the C12 mutations were required to maintain both its enhanced degradation and TMP stabilization. We further demonstrate the utility of C12 by fusing it to I{kappa}B and Nrf2, two stress-responsive proteins that have previously been challenging to regulate. In both instances, C12 significantly enhanced the basal turnover of these proteins and improved the dynamic range of regulation post stabilizer addition. These advantageous features of the C12 ecDHFR DD variant highlight its potential for replacing the conventional N-terminal ecDHFR DD, and overall improving the use of destabilizing domains, not only as a chemical biology tool, but for gene therapy avenues as well.

biochemistry↗

bioRxiv2022

Seed-competent tau monomer initiates pathology in PS19 tauopathy mice

Tau aggregation into ordered assemblies causes myriad neurodegenerative tauopathies. We previously reported that tau monomer exists in either inert (Mi) or seed-competent (Ms) conformational ensembles, and that Ms encodes strains, which are biologically active, self-propagating assemblies. We have previously isolated Ms from tauopathy brains, but it is unknown if disease begins with Ms formation followed by fibril assembly, or if Ms derives from fibrils and is an epiphenomenon. Consequently, we studied a tauopathy mouse model (PS19) that expresses full-length human (1N4R) tau containing a disease-associated mutation (P301S). Using tau repeat domain biosensor cells, we detected insoluble tau seeding activity at 2 months. We found insoluble tau protein assemblies by immunoblot at 3 months. We next immunopurified monomer from mice aged 1-6 weeks using size exclusion chromatography. We detected soluble seeding activity at 4 weeks, before insoluble material or larger assemblies, with assemblies ranging from n=1-3 tau units. By 5 and 6 weeks, large soluble assemblies had formed. This indicated the first detectable pathological forms of tau were Ms. We next tested for post-translational modifications of tau monomer from 1-6 weeks. We detected no phosphorylation unique to Ms in PS19 or Alzheimers disease brain. We conclude that tauopathy begins with formation of Ms monomer, whose activity is phosphorylation-independent. Ms self-assembles to form oligomers before it forms insoluble fibrils. The conversion of tau monomer from Mi to Ms thus constitutes the first detectable step in the initiation of tauopathy in this mouse model, with obvious implications for origins of disease in humans.

neuroscience↗

bioRxiv2021

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

biophysics↗