Biology subjects

Shahar, A.

Publications and source records attributed to Shahar, A..

7 recordsLinked to original sources

bioRxiv2026

Bovine coronavirus evolution preserves spike architecture while remodeling key functional regions

Bovine coronavirus (BCoV) is a major pathogen of cattle and the closest known ancestor of the human coronavirus OC43, yet how viral evolution reshapes spike protein structure and function remains poorly understood. Here, we combined comparative genomics, glycoproteomics, cryo-EM, and antigenic characterization to define the structural mechanisms underlying spike evolution across representative BCoV lineages. We identify a previously unrecognized lineage-specific N-glycosylation site in contemporary European viruses and validate its occupancy by glycoproteomics. High-resolution cryo- EM structures reveal that BCoV evolution preserves the overall prefusion architecture of the spike glycoprotein while selectively remodeling key functional regions involved in receptor recognition, conformational dynamics, and antigenicity. Comparative analysis with OC43 demonstrates increased conformational heterogeneity of the receptor-binding loop, whereas the Mebus vaccine strain exhibits enhanced membrane-proximal stalk flexibility despite maintaining thermal stability. Finally, structural modeling together with antibody-binding experiments reveals substantial antigenic remodeling despite >90% spike sequence identity between BCoV and OC43. Together, these findings establish a mechanistic framework for Embecovirus spike evolution and provide structural insights that may inform the development of vaccines based on contemporary circulating strains.

microbiology↗

bioRxiv2024

Privacy Preserving Epigenetic PaceMaker Stronger Privacy and Improved Efficiency

DNA methylation data plays a crucial role in estimating chronological age in mammals, offering real-time insights into an individuals aging process. The Epigenetic Pacemaker (EPM) model allows inference of the biological age as deviations from the population trend. Given the sensitivity of this data, it is essential to safeguard both inputs and outputs of the EPM model. In a recent study by Goldenberg et al., a privacy-preserving approach for EPM computation was introduced, utilizing Fully Homomorphic Encryption (FHE). However, their method had limitations, including having high communication complexity and being impractical for large datasets Our work presents a new privacy preserving protocol for EPM computation, analytically improving both privacy and complexity. Notably, we employ a single server for the secure computation phase while ensuring privacy even in the event of server corruption (compared to requiring two non-colluding servers in Goldenberg et al.). Using techniques from symbolic algebra and number theory, the new protocol eliminates the need for communication during secure computation, significantly improves asymptotic runtime and and offers better compatibility to parallel computing for further time complexity reduction. We have implemented our protocol, demonstrating its ability to produce results similar to the standard (insecure) EPM model with substantial performance improvement compared to Goldenberg et al. These findings hold promise for enhancing data security in medical applications where personal privacy is paramount. The generality of both the new approach and the EPM, suggests that this protocol may be useful to other uses employing similar expectation maximization techniques.

bioinformatics↗

bioRxiv2023

Acetylation-dependent coupling between G6PD activity and apoptotic signaling

Lysine acetylation has been discovered in thousands of non-histone human proteins, including most metabolic enzymes. Deciphering the functions of acetylation is key to understanding how metabolic cues mediate metabolic enzyme regulation and cellular signaling. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is acetylated on multiple lysine residues. Using site-specifically acetylated G6PD, we show that acetylation can activate (AcK89) and inhibit (AcK403) G6PD. Acetylation-dependent inactivation is explained by structural studies showing distortion of the dimeric structure and active site of G6PD. We provide evidence for acetylation-dependent K95/97 ubiquitylation of G6PD and Y503 phosphorylation, as well as interaction with p53 and induction of early apoptotic events. Notably, we found that the acetylation of a single lysine residue coordinates diverse acetylation-dependent processes. Our data provide an example of the complex roles of acetylation as a posttranslational modification that orchestrates the regulation of enzymatic activity, posttranslational modifications, and apoptotic signaling

biochemistry↗

bioRxiv2021

Neutralization of the anthrax toxin by antibody-mediated stapling of its membrane penetrating loop

Anthrax infection is associated with severe illness and high mortality. Protective antigen (PA) is the central component of the anthrax toxin, which is the main virulent factor of anthrax. Upon endocytosis, PA opens a pore in the membranes of endosomes, through which the toxins cytotoxic enzymes are extruded. The PA pore is formed by a cooperative conformational change where PAs membrane-penetrating loops associate, forming a hydrophobic rim that pierces the membrane. Due to its crucial role in anthrax progression, PA is an important target of monoclonal antibodies-based therapy. cAb29 is a highly effective neutralizing antibody against PA. We determined the cryo-EM structure of PA in complex with the Fab portion of cAb29. We found that cAb29 neutralizes the toxin by clamping the membrane-penetrating loop of PA to a static region on PAs surface, thereby preventing pore formation. Therefore, our results provide the structural basis for the antibody-based neutralization of PA and bring to focus the membrane-penetrating loop of PA as a target for the development of better anti-anthrax vaccines.

biochemistry↗

bioRxiv2020

The metal binding site composition of the cation diffusion facilitator protein MamM cytoplasmic domain impacts its metal responsivity

The cation diffusion facilitator (CDF) is a conserved family of divalent d-block metal cation transporters that extrude these cations selectively from the cytoplasm. CDF proteins are composed of two domains: the transmembrane domain, through which the cations are transported, and a regulatory cytoplasmic C-terminal domain (CTD). Metal binding to the CTD leads to its tighter conformation, and this sequentially promotes conformational change of the transmembrane domain which allows the actual transport of specific metal cations. It was recently shown that the magnetotactic bacterial CDF protein MamM CTD has a role in metal selectivity, as binding of different metal cations exhibits distinctive affinities and conformations. It is yet unclear whether the composition of the CTD binding sites can impact metal selectivity. Here we performed a mutational study of MamM CTD, where we exchanged the metal binding residues with different metal-binding amino acids. Using X-ray crystallography and Trp-fluorescence spectrometry, we studied the impact of the mutations on the CTD conformation in the presence of different metals. Our results reveal that the incorporation of such mutations alters the domain response to metals in vitro, as mutant forms of the CTD bind metals differently in terms of the composition of the binding sites and the CTD conformation. CoordinatesMamM CTD structures have been deposited in the Protein Data Bank under the following accession codes: 6H5V, 6H5M, 6H5U, 6H8G, 6HAO, 6H88, 6H87, 6H8A, 6H89, 6H8D, 6H5K, 6H9Q, 6H84, 6H83, 6HA2, 6H8I, 6H9T, 6H81, 6HAN, 6H85, 6H9P, 6HHS.

biophysics↗

bioRxiv2019

The inter-dimeric interface controls function and stability of Ureaplasma urealiticum methionine S-adenosyltransferase

Methionine S-adenosyltransferases (MATs) are predominantly homotetramers, comprised of dimers of dimers. The highly conserved dimeric interface harbors two active sites, making the dimer the obligatory functional unit. Yet, functionality of the recently evolved inter-dimeric interface remains unknown. Here, we show that the inter-dimeric interface of U. urealiticum MAT has evolved to control the catalytic activity and structural integrity of the homotetramer in response to product accumulation. When all four active sites are occupied with the product, S-adenosylmethionine (SAM), binding of four additional SAM molecules to the inter-dimeric interface prompts a [~]45{degrees} shift in the dimer orientation and a concomitant [~]60% increase in the interface area. This rearrangement inhibits the enzymatic activity by locking the flexible active site loops in a closed state and renders the tetramer resistant to proteolytic degradation. Our findings suggest that the inter-dimeric interfaces of MATs are recruited by evolution to tune the molecular properties of the entire homotetramer.

biochemistry↗

bioRxiv2019

Unexpected implications of STAT3 acetylation revealed by genetic encoding of acetyl-lysine

The signal transducer and activator of transcription 3 (STAT3) protein is activated by phosphorylation of a specific tyrosine residue (Tyr705) in response to various extracellular signals. STAT3 activity was also found to be regulated by acetylation of Lys685. However, the molecular mechanism by which Lys685 acetylation affects the transcriptional activity of STAT3 remains elusive. By genetically encoding the co-translational incorporation of acetyl-lysine into position Lys685 and co-expression with the Elk receptor tyrosine kinase, we were able to biochemically characterize site-specifically acetylated, and simultaneously acetylated and phosphorylated STAT3. We measured the effect of acetylation on the crystal structure, and DNA binding affinity and specificity of Tyr705-phosphorylated and non-phosphorylated STAT3. In addition, we monitored the deacetylation of acetylated Lys685 by reconstituting the mammalian enzymatic deacetylation reaction in live bacteria. Surprisingly, we found that acetylation, per se, had no effect on the crystal structure, and DNA binding affinity or specificity of STAT3, implying that the previously observed acetylation-dependent transcriptional activity of STAT3 involves an additional cellular component. In addition, we discovered that Tyr705-phosphorylation protects Lys685 from deacetylation in bacteria, providing a new possible explanation for the observed correlation between STAT3 activity and Lys685 acetylation.

biochemistry↗