Biology subjects

Zarivach, R.

Publications and source records attributed to Zarivach, R..

10 recordsLinked to original sources

bioRxiv2026

Genome degradation in the c-di-GMP signaling pathways and serovar-specific YhjH-YciG interactions rewired motility regulation in Salmonella Paratyphi A

Salmonella enterica serovars Typhimurium (STM) and Paratyphi A (SPA) cause clinically distinct diseases, yet the molecular bases for their different lifestyles remain incompletely understood. Genome degradation, a hallmark of typhoidal Salmonella, results in extensive pseudogenization across multiple functional pathways. Here, we investigate how such gene inactivation rewires cyclic-di-GMP (c-di-GMP) signaling and flagellar motility regulation in SPA vs. STM. We show that YhjH, a conserved phosphodiesterase (PDE), is required for motility in STM but not in SPA, despite retaining PDE activity in both serovars. We demonstrate that this functional divergence is caused by pseudogenization of ycgR in SPA, which truncates the flagellar brake protein YcgR to a nonfunctional peptide, severing the link between c-di-GMP levels and flagellar motor control. Site directed mutagenesis in the YhjH active site and ectopic expression of intact YcgR from STM that restored YhjH-dependent motility regulation in SPA, confirmed this molecular mechanism. Additionally, using a bacterial two-hybrid (BACTH) genetic screen, we identified a serovar-specific interaction between YhjH and the general stress protein YciG in SPA, but not in STM. Computational RNA folding analysis predicted substantial differences in mRNA secondary structure and stability between the SPA and STM yhjH alleles, suggesting a potential role for synonymous mutations in this serovar-specific interaction. Together, these findings reveal how genome degradation can rewire regulatory networks, uncovering a fundamental difference in motility control between typhoidal and non-typhoidal Salmonella and suggest that these differences allow SPA motility under conditions that suppress motility in STM. IMPORTANCESalmonella enterica serovars Typhimurium (STM) and Paratyphi A (SPA) cause fundamentally different diseases in humans, yet the molecular basis for their distinct lifestyles and pathogenicity remains poorly understood. Genome degradation is a hallmark of typhoidal Salmonella, but its functional consequences for regulatory networks are largely unexplored. Here, we demonstrate that pseudogenization of ycgR in SPA dismantles c-di-GMP-mediated flagellar motor control, liberating SPA from an inhibitory brake that suppresses motility in STM. Additionally, we uncover a serovar-specific interaction between the phosphodiesterase YhjH and the general stress protein YciG in SPA, demonstrating that genome degradation can drive regulatory network rewiring beyond simple gene loss. These differences in motility regulation may facilitate the systemic pathogenesis and unique lifestyle of SPA.

microbiology↗

bioRxiv2021

Asgard ESCRT-III and VPS4 reveal evolutionary conserved chromatin binding properties of the ESCRT machinery

The ESCRT machinery drive membrane remodeling in numerous processes in eukaryotes. Genes encoding for ESCRT proteins have been identified in Asgard archaea, a newly discovered superphylum, currently recognized as the ancestor of all eukaryotes. This begs the question of the functional evolutionary origin of this machinery and its conservation across lineages. Here, we find that Asgard-ESCRTs exhibit conserved DNA-binding properties, which is derived from recruitment of specific members. We show that Asgard-ESCRT-III/VPS4 homologs interact with one another inside mammalian cells, associate with chromatin, and recruit their counterparts to organize in discrete foci in the mammalian nucleus. This is congruent with human-ESCRT-III homologs. We find that human- and Asgard-ESCRT-IIIs associate with chromatin via the same N terminal domain, and that human-ESCRT-III can recruit Asgard-VPS4 to the nucleus to form foci. Therefore, ESCRTs possess chromatin binding properties that were preserved through the billion years of evolution that separate Asgard and human cells.

cell biology↗

bioRxiv2021

SAMase of bacteriophage T3 inactivates E. coli methionine S-adenosyltransferase by forming hetero-polymers

S-adenosylmethionine lyase (SAMase) of bacteriophage T3 degrades the intracellular SAM pools of the host E. coli cells, thus inactivating a crucial metabolite involved in plethora of cellular functions, including DNA methylation. SAMase is the first viral protein expressed upon infection and its activity prevents methylation of the T3 genome. Maintenance of the phage genome in a fully unmethylated state has a profound effect on the infection strategy -- it allows T3 to shift from a lytic infection under normal growth conditions to a transient lysogenic infection under glucose starvation. Using single-particle Cryo-EM and biochemical assays, we demonstrate that SAMase performs its function by not only degrading SAM, but also by interacting with and efficiently inhibiting the hosts methionine S-adenosyltransferase (MAT) -- the enzyme that produces SAM. Specifically, SAMase triggers open-ended head-to-tail assembly of E. coli MAT into an unusual linear filamentous structure in which adjacent MAT tetramers are joined together by two SAMase dimers. Molecular dynamics simulations together with normal mode analyses suggest that the entrapment of MAT tetramers within filaments leads to an allosteric inhibition of MAT activity due to a shift to low-frequency high-amplitude active site-deforming modes. The amplification of uncorrelated motions between active site residues weakens MATs ability to withhold substrates, explaining the observed loss of function. We propose that the dual function of SAMase as an enzyme that degrades SAM and as an inhibitor of MAT activity has emerged to achieve an efficient depletion of the intracellular SAM pools. IMPORTANCESelf-assembly of enzymes into filamentous structures in response to specific metabolic cues has recently emerged as a widespread strategy of metabolic regulation. In many instances filamentation of metabolic enzymes occurs in response to starvation and leads to functional inactivation. Here, we report that bacteriophage T3 modulates the metabolism of the host E. coli cells by recruiting a similar strategy -- silencing a central metabolic enzyme by subjecting it to phage-mediated polymerization. This observation points to an intriguing possibility that virus-induced polymerization of the host metabolic enzymes might be a common mechanism implemented by viruses to metabolically reprogram and subdue infected cells.

microbiology↗

bioRxiv2020

Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated heterozygous deletion

Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to alternating environments by stage-differentiation, accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression, since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E candidates, each assumed to have specific functions, although overlaps are expected. It is noted that some of them can bind to a dedicated eIF4G candidate partners, and LeishIF4E2 does not bind any known eIF4G ortholog. LeishIF4E2 was previously shown to comigrate in the polysomal fractions of sucrose gradients, whereas initiation factors usually comigrate with pre-initiation and initiation complexes. Using the CRISPR-Cas9 methodology, we deleted one of the two LeishIF4E2 gene copies. The deletion caused severe alterations in the morphology of mutant cells that turned round and equipped with a very short flagellum, but their growth rate and general translation remained unaffected. Proteomic analysis of the LeishIF4E2(+/-) mutant cells compared to wild type controls showed that the number of proteins that were upregulated exceeded the number of downregulated proteins, possibly indicating that a repressor function was eliminated. The upregulated proteins were related mainly to general metabolic processes, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included several groups, including cytoskeletal and ribosomal proteins. Despite the fact that only one of the two LeishIF4E2 alleles was deleted, the mutant cells were impaired in their ability to infect cultured macrophages. LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Although it could have a repressive function, we cannot exclude the possibility that it is responsible for translation of a specific set of transcripts. Overall, our results are in line with the possibility that the different LeishIF4Es are assigned unique functions. Author summaryLeishmania parasites cause a broad spectrum of diseases that lead to different pathological symptoms. During their life cycle, the parasites shuffle between sand-fly vectors and mammalian hosts, while adapting to changing environments via a stage specific program of gene expression that assists the survival of Leishmania under the changing conditions. Translation initiation plays a key role in control of gene expression, in Leishmania this is exemplified by the presence of multiple cap-binding complexes that interact with mRNAs. The parasites encode multiple paralogs of the cap-binding translation initiation factor eIF4E, and of its corresponding binding partner eIF4G, forming complexes with different potential functions. Using the CRISPR-Cas9 methodology, we generated a heterozygous mutant of the least studied cap-binding paralog, LeishIF4E2, eliminating one of its two alleles. The mutation caused morphological defects, resulting in short and rounded up cells with a significant reduction in their flagellar length. Although general translation rates and growth of the mutant parasite were not affected, total proteome analysis and translation assay suggested that LeishIF4E2 could possibly function as a translation repressor. Alternatively, LeishIF4E-2 could be responsible for promoting translation of a specific set of transcripts.

microbiology↗

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↗

bioRxiv2020

The cation diffusion facilitator protein MamM's cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2+

Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain (TMD), in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal, or multiple metals, from the cytoplasm. Here, the model CDF protein MamM, from magnetotactic bacteria, was used to probe the role of the CTD in metal selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of; their binding sites, thermodynamics and binding-dependent conformation, both in crystal form and in solution. Furthermore, the results indicate that the CTD discriminates against Mn2+ and provides the first direct evidence that CDF CTDs play a role in metal selectivity.

biophysics↗

bioRxiv2019

Integrative model to coordinate the oligomerization and aggregation mechanisms of CCL5

The CC-type chemokine ligand 5 (CCL5) is involved in the pathogenesis of many inflammatory conditions. The oligomerization and aggregation of CCL5 are considered to be responsible for its inflammatory properties. The CC-type dimer acts as the basic unit to constitute the oligomer. However, the structural basis of CCL5 oligomerization remains controversial. In this study, NMR and biophysical analyses proposed evidence that no single dimer-dimer interaction dominates in the oligomerization process of CCL5. CCL5 could oligomerize alternatively through two different interactions, E66-K25 and E66-R44/K45. In addition, a newly determined trimer structure reported an interfacial interaction through the N-terminal 12FAY14 sequence. The interaction contributes to aggregation and precipitation. In accordance with the observations, an integrative model explains the CCL5 oligomerization and aggregation process. CCL5 assembly consists of two types of dimer-dimer interactions and one aggregation mechanism. For full-length CCL5, the molecular accumulation triggers oligomerization through the E66-K25 interaction, and the 12FAY14 interaction acts as a secondary effect to derive aggregation. The E66-R44/K45 interaction dominates in CCL5 N-terminal truncations. The interaction would lead to filament-like formation in solution.

biochemistry↗

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↗

bioRxiv2017

Transition metal binding selectivity in proteins and its correlation with the phylogenomic classification of the cation diffusion facilitator protein family

Divalent d-block metal cations (DDMCs), such as Fe, Zn and Mn, participate in many biological processes. Understanding how specific DDMCs are transported to and within the cell and what controls their binding selectivity to different proteins is crucial for defining the mechanisms of metalloproteins. To better understand such processes, we scanned the RCSB Protein Data Bank, performed a de novo structural-based comprehensive analysis of seven DDMCs and found their amino acid binding and coordination geometry propensities. We then utilized these results to characterize the correlation between metal selectivity, specific binding site composition and phylogenetic classification of the cation diffusion facilitator (CDF) protein family, a family of DDMC transporters found throughout evolution and sharing a conserved structure, yet with different members displaying distinct metal selectivity. Our analysis shows that DDMCs differ, at times significantly, in terms of their binding propensities, and that in each CDF clade, the metal selectivity-related binding site has a unique and conserved sequence signature. However, only limited correlation exists between the composition of the DDMC binding site in each clade and the metal selectivity shown by its proteins.

biochemistry↗