Biology subjects

Shalev, D. E.

Publications and source records attributed to Shalev, D. E..

4 recordsLinked to original sources

bioRxiv2026

Site-specific phosphorylation of Ser352 drives aggregation of Tau R4 under acidosis conditions

In Alzheimers disease, neurons undergo acidosis as their pH drops from [~]7.1 to [~]6.5. Here we elucidate the molecular mechanism of specific Tau aggregation only at this lower pH. We show that a specific phosphorylation event in the Tau R4 domain is coupled to this pH drop to drive a defined phase transition from condensates to fibrils. Using a combination of experimental and computational studies, our results demonstrate that site-specific phosphorylation of Ser352 is the molecular switch that induces aggregation of Tau only at the more acidic, disease-related pH. We designed and synthesized a phosphopeptide library covering all phosphorylation patterns of the Tau-R4 domain and tested its response to controlled acidification using an array of complementary biophysical methods. This revealed that only a single phosphorylation of Ser352 led to acid-driven aggregation of Tau-R4. At neutral pH, Tau-R4 with a phosphorylation on Ser352 formed liquid condensates. These condensates converted irreversibly into amyloid filaments as the pH gradually decreased towards the level associated with pathological acidosis. Using a combination of 3{superscript 1}P-and 1H - NMR, MD simulations and microscopy studies, we show that the mechanism by which the phosphorylation of Ser352 Tau R4 exerts its effects is based on the unique position of this residue within the protein structure. pSer352 is located at the inside of the tip of a {beta}-hairpin, pointing into the hydrophobic core of the amyloid fold. This buried position increases its effective pK{square}, resulting in compacting of the hairpin following pSer352 protonation upon acidification. This shortens the inter-sheet distance at this position and tightens the filament. We conclude that this single protonation event of the pSer352 phosphate is responsible for the macroscopic phase transition from condensates to aggregates. Our results provide the molecular explanation for the specific aggregation of Tau under the pathologically relevant acidic pH, which is substantially different from its behavior at neutral pH.

biochemistry↗

bioRxiv2024

EspH utilizes phosphoinositide and Rab binding domains to interact with plasma membrane infection sites and Rab GTPases

Enteropathogenic E. coli (EPEC) is a Gram-negative bacterial pathogen that causes persistent diarrhea. Upon attachment to the apical plasma membrane of the intestinal epithelium, the pathogen translocates virulent proteins called effectors into the infected cells. These effectors hijack numerous host processes for the pathogens benefit. Therefore, studying the mechanisms underlying their action is crucial for a better understanding of the disease. We show that translocated EspH interacts with multiple host Rab GTPases. AlphaFold predictions and site-directed mutagenesis identified glutamic acid and lysine at positions 37 and 41 as Rab interacting residues. Mutating these sites abolished the EspH ability to inhibit the Akt and mTORC1 signaling, lysosomal exocytosis, and bacterial invasion. Knocking out the endogenous Rab8a gene expression highlighted the involvement of Rab8a in Akt/mTORC1 signaling and lysosomal exocytosis. A phosphoinositide binding domain with a critical tyrosine was identified in EspH. Mutating the tyrosine abolished the localization of EspH at infection sites and its capacity to interact with Rabs. Our data suggest novel EspH-dependent mechanisms that elicit immune signaling and membrane trafficking during EPEC infection.

cell biology↗

bioRxiv2021

Tandem WW/PPxY motif interactions in WWOX: the multifaceted role of the second WW domain

Class I WW domains mediate protein interactions by binding short linear PPxY motifs. They occur predominantly as tandem repeats, and their target proteins often contain multiple PPxY motifs, but the interplay of WW/peptide interactions is not always intuitive. WW domain-containing oxidoreductase (WWOX) protein harbors two WW domains: unstable WW1 capable of PPxY binding, and well-folded but mutated WW2 that cannot bind such motifs. WW2 is considered to act as a WW1 chaperone, but the underlying mechanism remains to be revealed. Here we combine NMR, ITC and structural modeling to elucidate the role of both WW domains in WWOX binding to single and double motif peptides derived from its substrate ErbB4. Using NMR we identified an interaction surface between the two domains that supports a WWOX conformation that is compatible with peptide substrate binding. ITC and NMR measurements reveal that while binding affinity to a single motif is marginally increased in the presence of WW2, affinity to a dual motif peptide increases tenfold, and that WW2 can directly bind double motif-peptides using its canonical binding site. Finally, differential binding of peptides in a mutagenesis study is consistent with a parallel orientation binding to the WW1-WW2 tandem domain, agreeing with structural models of the interaction. Our results reveal the complex nature of tandem WW domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both stability and binding. This opens the way to assess how evolution can utilize the multivariate nature of binding to fine-tune interactions for specific biological functions.

biophysics↗

bioRxiv2019

Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins

Formaldehyde is a widely used fixative in biology and medicine. The current mechanism of formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this mechanism. Instead, the data reveal that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield previously unrecognized fragments carrying a modification of one carbon atom. If these characteristics are considered, then formaldehyde cross-linking is readily applicable to the structural approach of cross-linking coupled to mass spectrometry. Using a cross-linked mixture of purified proteins, a suitable analysis identifies tens of cross-links that fit well with their atomic structures. A more elaborate in situ cross-linking of human cells in culture identified 469 intra-protein and 90 inter-protein cross-links, which also agreed with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. For example, two cross-links involving the protein {beta}NAC localize its binding site on the ribosome. Also of note are cross-links of actin with several auxiliary proteins for which the structure is unknown. Based on these findings we suggest a revised chemical reaction, which has relevance to the reactivity and toxicity of formaldehyde.

biochemistry↗