Biology subjects

Levin, L.

Publications and source records attributed to Levin, L..

13 recordsLinked to original sources

bioRxiv2026

The mitochondrial chaperone HSPD1 folds MTHFD2 independently of its co-chaperone HSPE1

Acquiring new cellular states entails metabolic reprogramming driven by changes in the expression of cytosolic and mitochondrial metabolic enzymes. Most mitochondrial proteins are synthesized in the cytosol and imported into the mitochondria in a linear form, after which they are folded by a network of mitochondrial chaperones and co-chaperones. Which mitochondrial protein is dependent upon which chaperone for its folding is largely unknown. HSPD1/HSPE1 (HSP60/HSP10) are evolutionarily conserved mammalian homologues of the bacterial proteins GroEL/GroES, forming a chamber-and-lid chaperonin to facilitate the folding of client proteins. We used gene knockdown and SILAC-based proteomics to identify HSPD1 client proteins. We found that HSPD1 supports the expression of Methylenetetrahydrofolate Dehydrogenase 2 (MTHFD2), a key essential protein in the mitochondrial one-carbon (1C) pathway, in cells and tumors, and directly folds MTHFD2, independently of its co-chaperone HSPE1. HSPD1 interacts with MTHFD2 in mitochondria, and MTHFD2 is degraded by LONP1 in HSPD1 knockdown cells. Consequently, we observed reduced nucleotide and S-adenosylmethionine (SAM) levels in HSPD1 knockdown and found minimal overlap in the transcriptional and metabolic cellular responses to HSPD1 vs. HSPE1 depletion. In C. elegans, knockout of HSP60 triggers the mitochondrial stress response in the gut, while HSP10 knockout triggers the mitochondrial stress response in muscle tissue. Our data support that HSPD1 is an MTHFD2 chaperone and that, in addition to working together, HSPD1 and HSPE1 have distinct biological functions.

cell biology↗

bioRxiv2026

Targeting CDK12/CYCLIN K induces HIV gene activation and latency reversal which is mediated by P-TEFb

The administration of antiretroviral therapy has successfully suppressed the replication of Human immunodeficiency virus and substantially inhibited viral infection. However, HIV still persists in long-lived infected cell reservoirs that are resistant to therapy and to immune clearance, therefore a barrier for elimination of infection. HIV gene expression is tightly regulated by the cellular transcription machinery, where low levels of host and viral transcription factors and epigenetic constrains maintain viral persistence. Here, we show that selective targeting of CDK12/CYCLIN K (CCNK) induces both HIV-specific and a global gene activation program, which is mediated by positive transcription elongation factor b (P-TEFb). Targeting CDK12/CCNK triggers a robust HIV gene expression, accompanied with latency reversal and synergistic reactivation when combined with latency-reversing agents. Mechanistically, CDK12/CCNK inhibition promotes the recruitment of RNA Polymerase II and CDK9 to the viral promoter, accompanied with elevated levels of histone activation makers. Targeting CDK12/ CCNK also exerts a global gene activation program, by releasing P-TEFb from 7SK snRNP, and reshaping the chromatin landscape at promoter-proximal loci and gene bodies. Together, these results uncover a previously unrecognized compensatory interplay between transcriptional kinases that rewires the cellular gene expression program with implications for HIV latency reversal. IMPORTANTO_LIManuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. C_LIO_LIUpon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. C_LIO_LIThe identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. C_LIO_LIThe identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. C_LI GUIDELINESO_LIFor reviewers: https://www.reviewcommons.org/reviewers C_LIO_LIFor authors: https://www.reviewcommons.org/authors C_LI CONTACTThe Review Commons office can be contacted directly at: office@reviewcommons.org

microbiology↗

bioRxiv2025

Germline stem cell isolation, lineage tracing, and aging in a protochordate

Germline stem cells (GSCs), the source of gametes, are the only stem cells capable of passing genes to future generations and are therefore considered units of natural selection. Yet, the factors that influence GSC fitness, and thus govern GSC competition, which exist in both protochordates and mammals, remain poorly understood. We studied how aging affects GSC fitness in the protochordate Botryllus schlosseri, an evolutionary crosspoint between invertebrates and vertebrates. GSCs were isolated and distinguished from developing and mature gametes using flow cytometry and scRNA-Seq, facilitated by a new PacBio genome assembly. Moreover, their function was validated through a novel lineage tracing approach that combines membrane-labeled GSC transplantation with scRNA-Seq. Leveraging our method to isolate them, single-cell transcriptomics showed significant age-related changes between young and old GSCs. Spermatids and sperm, however, showed minimal changes, suggesting that reproductive aging is governed by GSCs rather than by gametes. Reduced expressions of markers like DDX4 and PIWIL1 in aged GSCs mirrored trends in mammalian datasets, pointing to a conserved GSC-driven aging mechanism across chordate evolution. This study provides new techniques that lay the foundation to investigate further drivers of GSC fitness and highlights fertility-related genes as promising targets for therapies to preserve reproductive health.

cell biology↗

bioRxiv2025

Beyond translation initiation: dual regulatory interactions with phosphorylated and non-phosphorylated LeishIF4E1

Translation initiation in eukaryotes begins with the assembly of the cap-binding eIF4F complex at the 5 ends of mRNAs, through eIF4E. Among the six orthologs of eIF4E in Leishmania, LeishIF4E1 is intriguing, as it does not bind any Leishmania eIF4G. It is expressed in both life-forms, maintaining efficient cap-binding activity for translation initiation, unlike other LeishIF4Es. We identified two novel phosphorylation sites specifically S108, and S112, along with S203, which is conserved with the mammalian phosphorylation site. A non-phosphorylatable, tagged, mutant of LeishIF4E1 created by substituting the phosphorylated serine residues with alanine [S(108,112,203)A] was generated. The phosphorylation status did not affect the binding of LeishIF4E1 to translation initiation factors. However, the phosphorylated LeishIF4E1 interacted with proteins involved in DNA and chromatin structure, while non-phosphorylated LeishIF4E1 interacted with proteins that assist cells under stress and unfavorable conditions, particularly in gluconeogenesis. This suggests secondary regulatory roles for LeishIF4E1. RNA sequencing of the LeishIF4E1-associated transcripts revealed a tenfold reduction in transcripts binding to non-phosphorylated LeishIF4E1. GO enrichment analysis indicated distinct phosphorylation-dependent transcript specificities for each form. This study highlights the critical role of LeishIF4E1 phosphorylation in modulating the selectivity of protein interactome and transcript associations, extending the role of LeishIF4E1 beyond its well-established function in translation initiation. Author SummaryLeishmania are digenetic parasites that transition from sand fly vector to mammalian hosts, undergoing a developmental program of gene expression regulated at the level of translation. Protein synthesis is initiated by the assembly of multi-subunit complexes at the mRNA 5 cap structure and is recruited onto the ribosomes. There are six cap-binding proteins in Leishmania denoted LeishIF4Es1-6, and five LeishIF4G candidates. Among these, the function of LeishIF4E1 is intriguing, as it does not bind any LeishIF4G paralogs, and it is highly expressed in both parasite life forms. We examined how LeishIF4E1 phosphorylation modulates its interaction with proteins and transcripts, given that eIF4E phosphorylation in higher eukaryotes is known to regulate gene expression during malignancy. We identified three phosphorylation sites (S108, S112 and S203) and mutated them to A residues [S(108,112,203)A]. The mutated genes were transfected back into parasites for expression. Using pull-down assays, we identified the interacting proteins and associated transcripts for both the phosphorylated and non-phosphorylated LeishIF4E1 forms. The phosphorylated LeishIF4E1 pulled down proteins involved in DNA and chromatin structure, while the non-phosphorylated LeishIF4E1 interacted with proteins known to assist cells during stress and unfavorable conditions, especially gluconeogenesis. The transcript specificities differed markedly in the number of transcripts bound to both forms, shedding light on the importance of LeishIF4E1 phosphorylation. Furthermore, the phosphorylated form bound more transcripts encoding enzymes related to protein phosphorylation. We also showed that phosphorylation of LeishIF4E1 did not influence the cap-binding ability, as both the forms bind the m7GTP cap with parallel efficiencies. Our data suggest that the different cap-binding proteins in Leishmania are responsible for different processes and have a transcript-specific effect on gene expression.

microbiology↗

bioRxiv2025

SETD6 Mediates Selective Interaction and Genomic Occupancy of BRD4 and MITF in Melanoma cells

Aberrant transcriptional programs mediate malignant transformation of melanoma, the most aggressive form of skin cancer. The lysine methyltransferase SETD6 has been implicated in regulating transcription, cell adhesion, migration, and other processes in various cancers, however its role in melanoma remains unexplored. We recently reported that SETD6 mono-methylates the BRD4 at K99 to selectively regulate transcription of genes involved in mRNA translation. Here, we observed that BRD4 methylation at K99 by SETD6 occurs in melanoma cells. Knockout of SETD6 or a point mutation at BRD4-K99 disrupts BRD4 genomic occupancy. In addition, we show that SETD6 interacts with MITF, a master transcription factor in melanocytes and melanoma, and influences the genomic distribution of MITF. Mechanistically, we uncover a novel chromatin-localized interaction between BRD4 and MITF in melanoma. Our data suggest that BRD4 binds MITF in melanoma cells and that this interaction is dependent on both SETD6-mediated methylation of BRD4 and MITF acetylation. This chromatin complex plays a pivotal role in selective recruitment of BRD4 and MITF to different genomic loci in melanoma cells.

molecular biology↗

bioRxiv2025

Sperm meet the elevated energy demands to attain fertilization competence by increasing flux through aldolase

Prior to ejaculation, sperm are stored in the epididymis in a resting metabolic state. Upon ejaculation, sperm must alter their metabolism to generate the energy needed to support the motility and maturation process known as capacitation to reach and fertilize the oocyte. How sperm regulate the capacitation-induced increase in carbon flux is unknown. Here, we use 13C stable isotope labeling to follow glucose metabolism through sperm central carbon metabolic network before and after sperm activation. We identify regulatory steps which sperm use to alter their metabolic state from resting to highly active. In activated sperm, glucose flux through glycolysis is increased at the expense of the pentose phosphate pathway to increase energy yield. Increased glycolytic activity seems to be due to capacitation-induced stimulation of flux through aldolase. In the mitochondria-containing midpiece, glycolytically generated pyruvate feeds the TCA cycle to further maximize energy yield via oxidative phosphorylation. In the mitochondria-free principal piece of the tail, pyruvate produced from glycolysis is reduced to lactate by lactate dehydrogenase. Reduction to lactate regenerates oxidized NAD+ ensuring a sufficient supply to support glycolysis. The resultant lactate is at least partially secreted. Finally, we find evidence that there is an as yet unknown endogenous source of energy in sperm feeding the upregulation of TCA cycle intermediates. These studies provide the most complete picture of the metabolic shift which occurs in capacitating sperm. Significance statementA rapid switch from a quiescent to a high energy-demanding state during ejaculation is essential for sperm to reach and fertilize the oocyte. Somatic cells also undergo bioenergetic switches from low to very high energy demand. However, because metabolic processes essential for proliferation are going on in parallel, it is difficult to identify the molecular mechanisms regulating the increase in ATP production. This study represents the first complete picture of the metabolic reprogramming that happens in sperm upon ejaculation. Using stable isotope labeling, we identify rate-limiting enzymatic steps and points of regulation directing the changes in metabolic flux. Our sperm metabolic studies allow us to identify conserved mechanisms of metabolic regulation that are crucial for the survival of mammalian cells.

biochemistry↗

bioRxiv2025

Spatial constraints drive amylosome-mediated resistant starch degradation by Ruminococcus bromii in the human colon

Degradation of complex dietary fiber by gut microbes is essential for colonic fermentation, short-chain fatty acid production, and microbiome function. Ruminococcus bromii is the primary resistant starch (RS) degrader in humans, which relies on the amylosome, a specialized cell-bound enzymatic complex. To unravel its architecture, function, and the interplay among its components, we applied an holistic multilayered approach and found that amylosome composition RS degradation, and enzymatic synergy are regulated at two levels: structural constraints enforcing enzyme proximity and expression-driven shifts in enzyme proportions. Cryo-electron tomography revealed that the amylosome comprises a constitutive extracellular layer extending toward the RS. However, proteomics demonstrated its remodeling across different growth conditions, with Amy4 and Amy16 comprising 60% of the amylosome in response to RS. Structural and biochemical analyses revealed complementarity and synergistic RS degradation by these enzymes, which allow R. bromii to fine-tune its adaptation to dietary fiber and shape colonic metabolism.

microbiology↗

bioRxiv2024

A high-throughput zebrafish screen identifies novel candidate treatments for Kaposiform Lymphangiomatosis (KLA)

Kaposiform Lymphangiomatosis (KLA) is a rare, aggressive, and incurable disease caused by a somatic activating NRAS mutation (p.Q61R) in lymphatic endothelial cells (LECs). The development of new therapeutic avenues is hampered by the lack of animal models faithfully replicating the clinical manifestations of KLA. Here, we established a novel zebrafish model of KLA by driving conditional expression of the human NRAS mutation in venous and lymphatic ECs. We find that mutant embryos recapitulated clinical features of KLA, including pericardial edema and a dilated thoracic duct, and that the phenotypes were reverted by Trametinib, a MEK inhibitor used for KLA treatment. We further leverage this model in combination with an AI-based high-throughput drug screening platform to search for small compounds selectively reverting the mutant phenotypes and identify Cabozantinib, an FDA-approved tyrosine kinase inhibitor, and GSK690693, a competitive pan-Akt kinase inhibitor, as leading hits. Finally, we test these drugs in cultured cells derived from KLA patient and demonstrate their ability to normalize LEC sprouting and block NRAS downstream pathways, underscoring the potential of GSK690693 and Cabozantinib as potential KLA treatments. Overall, our novel zebrafish model provides a valuable tool for research into the etiology of KLA and for identifying new therapeutic avenues.

developmental biology↗

bioRxiv2023

Human subcutaneous and visceral adipocyte atlases uncover classical and specialized adipocytes and depot-specific patterns

Human adipose depots are functionally distinct. Yet, recent single-nucleus RNA-sequencing (snRNA-seq) analyses largely uncovered overlapping/similar cell-type landscapes. We hypothesized that adipocytes subtypes, differentiation trajectories, and/or intercellular communication patterns could illuminate this depot similarity-difference gap. For this, we performed snRNA-seq of human subcutaneous and visceral adipose tissue. Whereas the majority of adipocytes in both depots were classical, namely enriched in lipid metabolism pathways, we also observed specialized adipocyte subtypes that were enriched in immune-related, extracellular matrix deposition (fibrosis), vascularization/angiogenesis, or ribosomal processes. Pseudo-temporal analysis suggested a developmental trajectory from adipose progenitor cells to classical adipocytes via specialized adipocytes, suggesting that the classical state stems from loss, rather than gain, of specialized functions. Lastly, intercellular communication routes were consistent with the different inflammatory tone of the two depots. Jointly, these findings provide a high-resolution view into the contribution of cellular composition, differentiation, and intercellular communication patterns to human fat depot differences.

systems biology↗

bioRxiv2023

sNucConv: A bulk RNA-seq deconvolution method trained on single-nucleus RNA-seq data to estimate cell-type composition of human subcutaneous and visceral adipose tissues

Deconvolution algorithms rely on single-cell RNA-sequencing (scRNA-seq) data applied onto bulk RNA-sequencing (bulk RNA-seq) to extract information on the cell-types composition and proportions comprising a certain tissue. Adipose tissues cellular composition exhibits enormous plasticity in response to weight changes and high variance at different anatomical locations (depots). However, adipocytes - the functionally unique cell type of adipose tissue, are not amenable to scRNA-seq, a challenge recently met by applying single-nucleus RNA-sequencing (snRNA-seq). Here we aimed to develop a deconvolution method to estimate the cellular composition of human visceral and subcutaneous adipose tissues (hVAT and hSAT, respectively) using snRNA-seq to assess the true cell-type proportions. To correlate deconvolution-estimated cell-type proportions to true (snRNA-seq -derived) proportions, we analyzed seven hVAT and 5 hSAT samples by both bulk RNA-seq and snRNA-seq. snRNA-seq uncovered 15 distinct cell types in hVAT and 13 in hSAT. Deconvolution tools - SCDC, MuSiC, and Scaden exhibited low performance in estimating cell-type proportions (median |R|= 0.12 for estimated vs. true correlations). Notably, estimation accuracy somewhat improved by decreasing the number of cell-types groups, which nevertheless remained low (|R|<0.42). We therefore developed sNuConv, a novel method that employs Scaden, a deep-learning tool, trained using snRNA-seq - based data corrected by i. snRNA-seq/bulk RNA-seq highly-correlated genes, ii. corrected estimated cell-type proportions based on individual cell-type regression models. Applying sNuConv on our bulk RNA-seq data resulted in cell-type proportion estimation accuracy with median R=0.93 (range:0.76-0.97) for hVAT, and median R=0.95 (range:0.92-0.98) for hSAT. The resulting model was depot-specific, reflecting depot-differences in gene expression patterns. Thus, we present sNuConv, a novel, AI-based, method to deduce the cellular landscape of hVAT and hSAT from bulk RNA-seq data, providing proof-of-concept for producing validated deconvolution algorithms for tissues un-amenable to single-cell RNA sequencing.

bioinformatics↗

bioRxiv2021

Isolation and characterization of a highly specific monoclonal antibody targeting the botulinum neurotoxin type E exposed SNAP-25 neoepitope

Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. Specific detection and quantification of BoNT/E-cleaved SNAP-25 neoepitope is essential for diagnosis of BoNT/E intoxication as well as for characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. Immunized rabbits developed a specific and robust polyclonal antibody response, whereas immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. Sequence alignment of the isolated clones revealed high similarity between both heavy and light chains, with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled sensitive detection of the cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. This novel antibody can be further used to develop an in vitro cell-based assay to diagnose BoNT/E intoxication and to characterize antitoxin preparations, thus eliminating the use of animals in the standard mouse bioassay.

biochemistry↗

bioRxiv2021

External fertilization is orchestrated by a pH-regulated soluble adenylyl cyclase controlling sperm motility and chemotaxis

The reaction of CO2 with H2O to form HCO3- and H+ is one of the most important chemical equilibria in cells. In mammalian sperm, a soluble adenylyl cyclase (sAC) serves as cellular HCO3- sensor that conveys the equilibrium state via cAMP synthesis to cAMP-signaling molecules. The function of sAC and cAMP in non-mammalian sperm is largely unknown. Here, we identify sAC orthologs in sea urchin and salmon sperm that, surprisingly, are activated by alkaline pH rather than HCO3-. Two amino-acid residues required for HCO3- binding of mammalian sAC are lacking in pH-regulated sAC. Orthologs identified in ten other phyla are also lacking either one of these key residues, suggesting that pH control is widespread among non-mammalian metazoan. The pH-sensitive sAC controls several functions of sperm from external fertilizers. Upon spawning, alkalization triggers cAMP synthesis and, thereby, activates motility of quiescent sperm. Egg-derived chemoattractants also alkalize sperm and elevate cAMP, which then-modulates pacemaker HCN channels to trigger a chemotactic Ca2+ response. Finally, the sAC and the voltage- and cAMP-activated Na+/H+ exchanger sNHE mutually control each other. A picture of evolutionary significance is emerging: motility and sensory signaling of sperm from both internal and external fertilizers rely on cAMP, yet, their sAC is regulated by HCO3- or pHi, respectively. Acidification of aquatic habitats due to climate change may adversely affect pH-sensing by sAC and thereby sexual reproduction in the sea. Statement of significanceAdenylyl cyclases synthesize cAMP, a prominent cellular messenger. A bicarbonate-sensitive AC family member, soluble AC (sAC), is tied to the chemical equilibrium: H2O + CO2 {leftrightarrow} HCO3- (bicarbonate) + H+. The sAC is required for fertilization: Mammals lacking sAC are infertile and sperm immotile. We now identify a new sAC form in sperm of non-mammalian animals that reproduce in the sea. This novel sAC is activated at alkaline pH rather than bicarbonate. It controls sperm motility and chemotaxis. The switch from HCO3- to pH rests on substitution of two amino-acids, which represents an adaptation to aquatic environments low in bicarbonate. Acidification of aquatic habitats due to climate change may adversely affect sAC activity and, thereby, fertilization.

biophysics↗

bioRxiv2018

On the Development of Sesamoid Bones

Sesamoid bones are a special group of small auxiliary bones that form in proximity to joints and contribute to their stability and function. Sesamoid bones display high degree of variability in size, location, penetrance and anatomical connection to the main skeleton across vertebrate species. Therefore, providing a comprehensive developmental model or classification system for sesamoid bones is challenging. Here, we examine the developmental mechanisms of three anatomically different sesamoid bones, namely patella, lateral fabella and digit sesamoids. Through a comprehensive comparative analysis at the cellular, molecular and mechanical levels, we demonstrate that all three types of sesamoid bones originated from Sox9+/Scx+ progenitors under the regulation of TGF{beta} and independent of mechanical stimuli from muscles. We show that BMP4 was necessary specifically for differentiation of patella but not of lateral fabella or digit sesamoids, whereas BMP2 regulated the growth of all examined sesamoids. Next, we show that whereas patella and digit sesamoids initially formed in juxtaposition to long bones, the lateral fabella formed independently at a distance. Finally, we provide evidence suggesting that while patella detached from the femur by formation of a synovial joint, digit sesamoids detached from the phalanx by a fibrocartilage joint. Collectively, these findings highlight both common and divergent molecular and mechanical features of sesamoid bone development, thereby advancing our understanding of their evolutionary plasticity.

developmental biology↗