Biology subjects

Levin, T.

Publications and source records attributed to Levin, T..

8 recordsLinked to original sources

bioRxiv2025

Differential targeting of human pyroptotic Caspase-5 and Caspase-4 by Shigella OspC2 and OspC3

Pyroptosis is an inflammatory cell death pathway that is a key defense mechanism of intestinal epithelial cells. To successfully establish an infection, intracytosolic Gram-negative pathogens must block this host response. Indeed, Shigella OspC3 suppresses epithelial pyroptosis by targeting and inactivating Caspase-4 (CASP4). Here, we demonstrate that OspC2, which shares 96% identity with OspC3, targets Caspase-5 (CASP5), a close paralog of CASP4. Through a combination of yeast two-hybrid, transfection, and bacterial infection assays, we show that the distinct pyroptotic caspase specificities of OspC2 and OspC3 are determined by a short -helical region, designated the Pyroptotic Caspase Specificity (PCS) domain. This domain is located upstream from the Ankyrin Rich Repeat (ARR) region previously established to promote OspC3 binding to CASP4. Swapping PCS domains between OspC2 and OspC3 is sufficient to redirect their caspase targeting. Evidence for CASP5-driven pyroptosis in response to infection has not yet been established. However, CASP5 displays signatures of positive selection at residues predicted to interact with the PCS domain of OspC2. Notably, the introduction of orangutan-specific residues into human CASP5 disrupt its interaction with and modification by OspC2, demonstrating that CASP5 natural variation can cause key functional differences in this host-microbe molecular interaction. These findings highlight the evolutionary interplay between bacterial effectors and host proteins, support a likely role for CASP5 in responding to Gram-negative bacteria, and identify promising therapeutic targets for enhancing epithelial defense against bacterial pathogens. ImportanceShigella species are human-specific Gram-negative pathogens that establish a replicative niche in intestinal epithelial cells by blocking pyroptosis, a key inflammatory cell death pathway. We reveal that two closely related Shigella type III secreted effectors, OspC2 and OspC3, specifically inactivate the human caspase paralogs CASP5 and CASP4, respectively. This specificity is determined by their newly identified Pyroptotic Caspase Specificity (PCS) domains. In addition, we find that positively selected residues in CASP5 alter the OspC2/CASP5 interaction, underscoring the impacts of ongoing evolutionary arms races between bacterial effectors and host immune proteins. By elucidating the molecular basis of caspase targeting and adaptation, this work provides new insight into the diversification of host defense mechanisms and identifies potential therapeutic targets for enhancing epithelial resistance to bacterial infection.

microbiology↗

bioRxiv2025

Hypermutable hotspot enables the rapid evolution of self/non-self recognition genes in Dictyostelium

Cells require highly polymorphic receptors to perform accurate self/non-self recognition. In the amoeba Dicytostelium discoideum, polymorphic TgrB1 & TgrC1 proteins are used to bind sister cells and exclude cheaters, but it remains unknown how cells continually generate this extreme genetic diversity. Here, we created a collection of chromosome-length, whole genome sequences from 10 Dictyostelium discoideum isolates and sister species to understand the evolution of the large tgr gene family. Our dataset includes AX2-214, a widely used D. discoideum lab strain, as well as complete genomes for two Chlamydia-like endosymbionts harbored within amoebae. We find that tgrB1 and C1 lie in a hypermutational hotspot, with haplotypes that undergo repeated intralocus recombination, duplications, transpositions, and inversions. These structural dynamics are highly localized adjacent to tgrB and C, resulting in the gain and loss of dozens of genes. The tgrBC genes themselves frequently duplicate and recombine, leading to the rapid generation of unique tgrBC repertoires. In the broader tgr gene family, some genes (e.g. tgrN) are single copy and syntenic across all the genomes, whereas others (e.g. tgrA) prolifically duplicate at similar rates to Dictyostelium transposons. Thus, the tgr genes are among the most rapidly evolving families genome-wide. We propose that the intense diversification within the tgrBC locus can help explain how these genes acquire such extreme levels of polymorphism, with parallels to the MHC immune genes in mammals and other allorecognition systems. This collection of amoeba genomes is also an ideal dataset for comparative genomics and molecular evolution in Amoebozoa. SignificanceThe ability to distinguish self from non-self is an essential part of innate immunity and multicellularity. In the amoeba Dicytostelium discoideum, cells form transient multicellular structures via aggregation. In the process, they exclude distantly-related cheater strains via highly polymorphic cell surface proteins, TgrB1 and TgrC1. We used this system to ask: how do organisms continually generate new variation in recognition factors? After sequencing a collection of Dictyostelium spp. genomes, we found that the tgrB and tgrC genes lie in an extraordinarily variable locus- a region with such high rates of gene birth and death that genomic similarities are quickly lost, even between closely related isolates. Thus, amoeba cell recognition mirrors self/non-self evolutionary dynamics found across the Tree of Life.

evolutionary biology↗

bioRxiv2024

Profiling the physiological impact of aberrant folded-state protein filamentation in cells

The formation of large polymeric structures such as cytoskeletal and enzyme filaments is crucial for normal cellular function. However, such filaments can also form due to mutations that create self-interactions at the surface of symmetric proteins. Often, the proteins forming these structures maintain a folded state and thereby differ from aggregates and amyloids that involve misfolding. We refer to this type of assemblies as agglomerates to mark this difference. While cells have quality control mechanisms to identify, buffer, and eliminate misfolded proteins, it is unclear whether similar mechanisms exist for agglomerates, or whether agglomerates are toxic to cells. Here, we profiled the physiological impact of mutation-induced folded-state protein filamentation in yeast cells. First, we devised a simple strategy to distinguish fluorescently labeled proteins forming agglomerates versus aggregates. We then profiled exogenous protein agglomerates in terms of their recognition by known quality control mechanisms, their effects on specific cellular processes and overall fitness on S. cerevisiae cultures. We found that agglomerates do not colocalize with the proteostasis machinery and do not result in measurable fitness defects. Proteomics profiling of cells expressing the wild type protein, agglomerating or misfolded variants revealed a consistent picture, with only minor, agglomerate-size-dependent changes observed and linked to the cell-wall and plasma-membrane proteins. Overall, our findings indicate that agglomerates form mostly benign structures in cells when compared to aggregates, and thereby offer a promising route for synthetic biology applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/612878v1_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@7136a4org.highwire.dtl.DTLVardef@ba62dborg.highwire.dtl.DTLVardef@b02bbaorg.highwire.dtl.DTLVardef@6cbf9_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Evolutionary dynamics of pro-inflammatory caspases in primates and rodents

Caspase-1 and related proteases are key players in inflammation and innate immunity. Here, we characterize the evolutionary history of caspase-1 and its close relatives across 19 primates and 21 rodents, focusing on differences that may cause discrepancies between humans and animal studies. While caspase-1 has been retained in all these taxa, other members of the caspase-1 subfamily (caspase-4, -5, -11, -12, and CARD16, 17, and 18) each have unique evolutionary trajectories. Caspase-4 is found across simian primates, whereas we identified multiple pseudogenization and gene loss events in caspase-5, caspase-11, and the CARDs. Because caspases-4 and -11 are both key players in the non-canonical inflammasome pathway, we expected that these proteins would be likely to evolve rapidly. Instead, we found that these two proteins are largely conserved, whereas caspase-4s close paralog, caspase-5, showed significant indications of positive selection, as did primate caspase-1. Caspase-12 is a non-functional pseudogene in humans. We find this extends across most primates, although many rodents and some primates retain an intact, and likely functional, caspase-12. In mouse laboratory lines, we found that 50% of common strains carry non-synonymous variants that may impact the functions of caspase-11 and -12, and therefore recommend specific strains to be used (and avoided). Finally, unlike rodents, primate caspases have undergone repeated rounds of gene conversion, duplication, and loss leading to a highly dynamic pro-inflammatory caspase repertoire. Thus we uncovered many differences in the evolution of primate and rodent pro-inflammatory caspases, and discuss the potential implications of this history for caspase gene functions.

evolutionary biology↗

bioRxiv2023

An atlas of protein homo-oligomerization across domains of life

Protein structures are essential to understand cellular processes in molecular detail. While advances in AI revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. Here, we describe a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life. We find that 50% of archaeal, 45% of bacterial, and 20% of eukaryotic proteomes form homomers. Our predictions accurately capture protein homo-oligomerization, recapitulate megadalton complexes, and unveil hundreds of novel homo-oligomer types. Analyzing these datasets reveals coiled-coil regions as major enablers of quaternary structure evolution in Eukaryotes. Integrating these structures with omics data shows that a majority of known protein complexes are symmetric. Finally, these datasets provide a structural context for interpreting disease mutations, which we find enriched at interfaces. Our strategy is applicable to any organism and provides a comprehensive view of homo-oligomerization in proteomes, protein networks, and disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/544317v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@1507a12org.highwire.dtl.DTLVardef@7e522aorg.highwire.dtl.DTLVardef@1445410org.highwire.dtl.DTLVardef@eb09f7_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

L. pneumophila resists its self-harming metabolite HGA via secreted factors and collective peroxide scavenging

Many pathogenic bacteria, including Legionella pneumophila, infect humans from environmental reservoirs. To survive in these reservoirs, bacteria must withstand microbe-on-microbe competition. We previously discovered that L. pneumophila can compete with neighboring bacteria via an antimicrobial metabolite called homogentisic acid (HGA) (Levin, Goldspiel, and Malik 2019). Curiously, L. pneumophila strains that secrete HGA are not wholly immune to its effects: low-density bacteria are strongly inhibited by HGA whereas high-density cells are tolerant. How do these bacteria tolerate HGA and avoid self-harm during interbacterial competition? Here, we find that HGA toxicity occurs via the production of toxic hydroperoxides and multiple factors facilitate high-density tolerance. First, HGA only becomes fully toxic after >1 hour of oxidation. While this manifests as a delay in killing within well-mixed liquid cultures, in a biofilm environment, this could provide time for HGA to diffuse away before becoming toxic. Second, HGA generates quantities of hydroperoxides that can be collectively scavenged by high-density, but not low-density cells. And third, high-density cells produce one or more secreted factors that are transiently protective from HGA. In combination, we propose that the bacteria are able to deploy HGA to generate a pool of reactive oxygen species surrounding their own biofilms, while maintaining non-toxic conditions within them. Overall, these findings help to explain how broadly toxic molecules can be used as inter-bacterial weapons. They also provide insights about why some of our current decontamination methods to control L. pneumophila are ineffective, leading to recurrent disease outbreaks.

microbiology↗

bioRxiv2021

Mutants libraries reveal negative design shielding proteins from mis-assembly and re-localization in cells

Understanding the molecular consequences of mutations in proteins is essential to map genotypes to phenotypes and interpret the increasing wealth of genomic data. While mutations are known to disrupt protein structure and function, their potential to create new structures and localization phenotypes has not yet been mapped to a sequence space. To map this relationship, we employed two homo-oligomeric protein complexes where the internal symmetry exacerbates the impact of mutations. We mutagenized three surface residues of each complex and monitored the mutations effect on localization and assembly phenotypes in yeast cells. While surface mutations are classically viewed as benign, our analysis of several hundred mutants revealed they often trigger three main phenotypes in these proteins: nuclear localization, the formation of puncta, and fibers. Strikingly, more than 50% of random mutants induced one of these phenotypes in both complexes. Analyzing the mutants sequences showed that surface stickiness and net charge are two key physicochemical properties associated with these changes. In one complex, more than 60% of mutants self-assembled into fibers. Such a high frequency is explained by negative design: charged residues shield the complex from misassembly, and the sole removal of the charges induces its assembly. A subsequent analysis of several other complexes targeted with alanine mutations suggested that negative design against mis-assembly and mislocalization is common. These results highlight that minimal perturbations in protein surfaces physicochemical properties can frequently drive assembly and localization changes in a cellular context.

biophysics↗

bioRxiv2018

Glycosyltransferase homologs prevent promiscuous cell aggregation and promote multicellular development in the choanoflagellate S. rosetta

The mechanisms underlying multicellular development in the animal stem lineage may be reconstructed through the study of choanoflagellates, the closest living relatives of animals. To determine the genetic underpinnings of multicellularity in the emerging model choanoflagellate S. rosetta, we performed a screen for mutants with defects in multicellular rosette development. In two of the mutants, Jumble and Couscous, single cells failed to develop into orderly rosettes but instead aggregated promiscuously into amorphous clumps of cells. Both mutants mapped to lesions in genes encoding glycosyltransferases and the mutations perturbed glycosylation patterns in the extracellular matrix (ECM). In animals, glycosyltransferases transfer activated sugars to donor molecules and thereby sculpt the polysaccharide-rich ECM, regulate integrin and cadherin activity, and, when disrupted, contribute to tumorigenesis. The finding that glycosyltransferases promote proper rosette development and prevent cell aggregation in S. rosetta suggests a pre-metazoan role for glycosyltransferases in regulating development and preventing abnormal tumor-like multicellularity.\n\nIMPACT STATEMENTA genetic screen reveals that glycosyltransferases are required for proper rosette development and the prevention of cell clumping in one of the closest living relatives of animals, the choanoflagellate S. rosetta.

genetics↗