Biology subjects

Savidor, A.

Publications and source records attributed to Savidor, A..

14 recordsLinked to original sources

bioRxiv2025

Two opposing redox signals mediated by 2-Cys peroxiredoxin shape the redox proteome during photosynthetic induction

Photosynthetic induction, characterized by the lag in CO2 assimilation rates typically observed upon plant transition from darkness to light, has traditionally been attributed to Rubisco activase activity and stomatal opening. Yet, the faster induction of photosynthesis in the 2-Cys peroxiredoxins (Prxs) mutant (2cpab) highlighted the critical role of chloroplast redox state in regulating photosynthetic rates during this phase. Since 2-Cys Prxs are involved in transmission of oxidative signals to target enzymes, it was hypothesized that it slows down photosynthesis during the induction phase. SPEAR, a redox proteomics approach for simultaneous protein expression and redox analysis, was used to systematically map redox changes occurring at the proteome level during photosynthesis induction and to unravel the role of 2-Cys Prxs in shaping these redox alterations. No significant difference was observed in protein expression levels between WT and 2cpab plants, suggesting that protein abundance does not account for the 2cpab phenotype. During the transition from dark to low light, 82 and 54 cysteine-containing peptides were reduced or oxidized, respectively, in WT plants. Most redox-regulated cysteines in photosynthetic proteins were found oxidized in the dark and became reduced in response to light, including ATP synthase gamma chain 1 (ATPC1) and glyceraldehyde-3-phosphate dehydrogenase (GAPB). A reverse pattern was observed among redox-regulated cysteines in proteins involved in starch degradation and chloroplast glycolysis, which shifted from a reduced to an oxidized state in response to light. These findings demonstrate the initiation of two opposing redox responses, affecting distinct sets of metabolic proteins during the induction phase. Remarkably, a significantly lower number of cysteines were reduced or oxidized in 2cpab plants, highlighting the crucial role 2-Cys Prxs play in shaping both signals. Taken together, rotational shifts between metabolic pathways during the photosynthesis induction phase are regulated by two opposing redox signals mediated by 2-Cys Prx activity.

plant biology↗

bioRxiv2025

ELIP is preferentially expressed in stomatal guard cells and has a role in stomatal opening upon transition to light

Plant early light-induced proteins (ELIPs) are chlorophyll-binding thylakoid membrane proteins commonly believed to function in photoprotection. However, results of studies with mutants or transgenic plants have been contradictory regarding this function, and the mechanistic workings of ELIPs have mostly remained elusive. We studied: (1) ELIP function in tomato (Solanum lycoperiscum) by generation and photosynthetic/physiological characterization of slelip mutants under various light conditions; and (2) ELIP tissue/cell localization by monitoring expression of the Venus yellow fluorescent protein fused to the native SlELIP1 promoter ( pSlELIP1::Venus) in transgenic tomato. Notably, slelip mutants did not display compromised photosynthetic performance and were not more sensitive to photoinhibition compared to parental plants, even following direct sunlight exposure. Surprisingly, pSlELIP1::Venus fusions revealed preferential expression in the leaf epidermis and specifically in stomatal guard cells, with no apparent expression in the mesophyll. No major alterations were observed in leaf gas exchange in slelip mutants at different light conditions. Intriguingly, stomatal conductance was reduced in slelip mutants upon the transition from dark to light. We propose an alternative hypothesis for plant ELIPs as players in the response of guard cells to light, connecting chlorophyll to stomatal function, and presenting a novel research direction for these enigmatic proteins.

plant biology↗

bioRxiv2024

The RNA-binding protein PRRC2B preserves 5' TOP mRNA during starvation to maintain ribosome biogenesis during nutrient recovery

PRRC2B is an intrinsically disordered RNA-binding protein that is part of the cells translation machinery. Here we show that PRRC2B has two alternatively spliced mRNA transcripts producing major long and minor short isoforms. Mass spectrometry-based interaction studies indicated that both isoforms associate with the 40S ribosomal subunit and translation initiation factors. Importantly, the long isoform also interacted with additional RNA-binding proteins through its unique Arg/Gly-rich region. Among these is LARP1, a regulator of 5 terminal oligopyrimidine (TOP) mRNAs under conditions of mTOR inhibition. We discovered that like LARP1, PRRC2B is necessary for preservation of 5 TOP mRNA levels, particularly those encoding ribosomal proteins, during amino acid starvation. In its absence, the rapid de novo translation of ribosomal proteins that takes place upon nutrient recovery is impeded. Overall, our study elucidates a newly discovered function for PRRC2B as an RNA-binding protein that regulates ribosomal biogenesis upon metabolic shift, in addition to its established function in initiating translation of specific mRNA targets.

molecular biology↗

bioRxiv2024

TIR signaling activates caspase-like immunity in bacteria

Proteases of the caspase family, as well as Toll/Interleukin-1 Receptor (TIR)-domain proteins, have central roles in innate immunity and regulated cell death in humans. In this study we describe a bacterial immune system comprising both a caspase-like protease and a TIR-domain protein. We found that the TIR protein, once it recognizes phage invasion, produces the previously unknown immune signaling molecule ADP-cyclo[N7:1'']-ribose (N7-cADPR). This molecule specifically activates the bacterial caspase-like protease which then indiscriminately degrades cellular proteins to halt phage replication. The TIR-caspase defense system, which we denote as type IV Thoeris, is abundant in bacteria and efficiently protects against phage propagation. Our study highlights the diversity of TIR-produced immune signaling molecules and demonstrates that cell death regulated by proteases of the caspase family is an ancient mechanism of innate immunity.

microbiology↗

bioRxiv2024

Altered Extracellular Matrix Structure and Elevated Stiffness in a Brain Organoid Model for Disease

The viscoelasticity of tissues impacts their shape, as well as the growth and differentiation of their cells. Nevertheless, little is known about changes in viscoelastic properties during brain malformations. Lissencephaly is a severe malformation of cortical development caused by LIS1 mutations, which results in a lack of cortical convolutions. Here, we show that human-derived brain organoids with LIS1 mutation are stiffer than control ones at multiple developmental times. This stiffening is accompanied by abnormal ECM expression and organization, as well as elevated water content, as measured by diffusion-weighted MRI. Proteolytic cleavage of ECM components by short-term treatment with the catalytic subunit of MMP9 reduced the stiffening and water diffusion levels of mutated organoids to control levels. Finally, based on the molecular and rheological properties obtained, we generated a computational microstructure mechanical model that can successfully predict mechanical changes that follow differential ECM localization and integrity in the developing brain. Overall, our study reveals that LIS1 is essential for the expression and organization of ECM proteins during brain development, and its mutation leads to a substantial viscoelastic change. To our knowledge, this is the first study to elucidate how tissue mechanics change in disease states using human brain organoids.

developmental biology↗

bioRxiv2023

Loss of EIF4G2 Mediates Aggressiveness in Distinct Human Endometrial Cancer Subpopulations with Poorer Survival Outcome in Patients

The non-canonical translation initiation factor EIF4G2 plays essential roles in embryonic development and differentiation, and contributes to the cellular stress response via translation of selective mRNA cohorts. Currently there is limited and conflicting information regarding the potential involvement of EIF4G2 in cancer development and progression. Endometrial cancer (EC) is the most pervasive gynecological cancer in the developed world, with increasing incidence every year. High grade ECs are largely refractory to conventional treatments, presenting poor survival rates and lacking suitable prognostic markers. Here we assayed a cohort of 280 EC patients across different types, grades, and stages, and found that low EIF4G2 expression highly correlated with poor overall and recurrence free survival in Grade 2 EC patients, monitored over a period of up to 12 years. To establish a causative connection between low EIF4G2 expression and cancer progression, we analyzed in parallel two independent human EC cell lines and demonstrated that stable EIF4G2 knock-down resulted in increased resistance to conventional therapies. Depletion of EIF4G2 also increased the prevalence of molecular markers for aggressive cell subsets, and altered their transcriptional and proteomic landscapes. Prominent among the proteins with decreased abundance were Kinesin-1 motor proteins KIF5B and KLC1, 2, 3. Multiplexed imaging of the tumors from this EC patient cohort showed a correlation between decreased protein expression of either KIF5B or KLC1, and poor survival in patients of certain grades and stages. The findings herein reveal potential novel biomarkers for Grade 2 EC with potential ramifications for patient stratification and therapeutic interventions. SignificanceDecreased EIF4G2 protein results in increased drug resistance of aggressive sub-populations of endometrial cancer cells, is associated with poor patient survival, and may serve as a novel prognosis marker for endometrial cancer.

cancer biology↗

bioRxiv2023

Loss-of-function cancer-associated mutations in the EIF4G2 non-canonical translation initiation factor

Tumor cells often exploit the protein translation machinery, resulting in enhanced protein expression essential for tumor growth. Since canonical translation initiation is often suppressed due to cell stress in the tumor microenvironment, non-canonical translation initiation mechanisms become particularly important for shaping the tumor proteome. EIF4G2 is a non-canonical translation initiation factor that mediates internal ribosome entry site [IRES] and upstream open reading frame [uORF] dependent initiation mechanisms, which can be used to modulate protein expression in cancer. Here we explored the contribution of EIF4G2 to cancer by screening the COSMIC database for EIF4G2 somatic mutations in cancer patients. Functional examination of missense mutations revealed deleterious effects on EIF4G2 protein-protein interactions, and importantly, on its ability to mediate non-canonical translation initiation. Specifically, one mutation, R178Q, led to reductions in protein expression and near complete loss-of-function. Two other mutations within the MIF4G domain specifically affected EIF4G2s ability to mediate IRES-dependent translation initiation but not that of target mRNAs with uORFs. These results shed light on both the structure-function of EIF4G2 and its potential tumor suppressor effects.

cancer biology↗

bioRxiv2022

Redifferentiated cardiomyocytes retain residual dedifferentiation signatures and are protected against ischaemic injury

Cardiomyocyte renewal by dedifferentiation and proliferation has fueled the field of regenerative cardiology in recent years, while the reverse process of redifferentiation remains largely unexplored. Redifferentiation is characterised by the restoration of function that is lost during dedifferentiation and is key to the healing process following injury. Previously, we showed that ERBB2-mediated heart regeneration has these two distinct phases: dedifferentiation, followed by redifferentiation. Here, using temporal RNAseq and proteomics, we survey the landscape of the dedifferentiation-redifferentiation process in the adult mouse heart. We find well characterised dedifferentiation pathways, such as reduced oxphos, increased proliferation and increased EMT-like features, largely return to normal, though elements of residual dedifferentiation remain, even after contractile function is restored. These hearts appeared rejuvenated and showed robust resistance to ischaemic injury. We find that redifferentiation is driven by negative feedback signalling, notably through LATS1/2 Hippo pathway activity. Disabling LATS1/2 in dedifferentiated cardiomyocytes augments dedifferentiation in vitro and prevents redifferentiation in vivo. Taken together, our data reveal the non-trivial nature of redifferentiation, whereby elements of dedifferentiation linger in a surprisingly beneficial manner. This cycle of dedifferentiation-redifferentiation protects against future insult, in what could become a novel prophylactic treatment against ischemic heart disease for at-risk patients.

cell biology↗

bioRxiv2021

Widespread Utilization of Diverse Organophosphate Pollutants by Marine Bacteria

Anthropogenic organophosphates (AOPs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents and pesticides. Soil bacteria bearing a phosphotriesterase (PTE) can degrade AOPs, but whether bacteria are capable of utilizing AOPs as a phosphorus source, and how widespread PTEs are in nature, remains unclear. Here, we report the utilization of diverse AOPs by four model marine bacteria and seventeen bacterial isolates from seawater samples. To unravel the details of AOP utilization, two novel PTEs from marine bacteria were isolated and characterized. When expressed in E. coli, these PTEs enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPs provides bacteria with a source of phosphorus in depleted environments and offers a new prospect for the bioremediation of a pervasive class of anthropogenic pollutants. One sentence summaryWidespread utilization of diverse organophosphate pollutants by over 20 marine bacterial strains represents a new hope for ocean bioremediation.

microbiology↗

bioRxiv2021

SPEAR: a proteomics approach for simultaneous protein expression and redox analysis

Oxidation and reduction of protein cysteinyl thiols serve as molecular switches, which is considered the most central mechanism for redox regulation of biological processes, altering protein structure, biochemical activity, subcellular localization, and binding affinity. Redox proteomics allows for the global identification of redox-modified cysteine (Cys) sites and quantification of their oxidation/reduction responses, serving as a hypothesis-generating platform to stimulate redox biology mechanistic research. Here, we developed Simultaneous Protein Expression and Redox (SPEAR) analysis, a new redox-proteomics approach based on differential labeling of oxidized and reduced cysteines with light and heavy isotopic forms of commercially available isotopically-labeled N-ethylmaleimide (NEM). The presented method does not require enrichment for labeled peptides, thus enabling simultaneous quantification of Cys oxidation state and protein abundance. Using SPEAR, we were able to quantify the in-vivo oxidation state of thousands of cysteines across the Arabidopsis proteome under steady-state and oxidative stress conditions. Functional assignment of the identified redox-sensitive proteins demonstrated the widespread effect of oxidative conditions on various cellular functions and highlighted the enrichment of chloroplast-targeted proteins. SPEAR provides a simple, straightforward, and cost-effective means of studying redox proteome dynamics. The presented data provide a global quantitative view of cysteine oxidation of well-known redox-regulated active sites and many novel redox-sensitive sites whose role in plant acclimation to stress conditions remains to be further explored.

biochemistry↗

bioRxiv2021

The Serine Protease HtrA2 mediates radiation-induced senescence in cancer cells

Radiation therapy can induce cellular senescence in cancer cells leading to short-term tumor growth arrest, yet increased long-term recurrence. To better understand the molecular mechanisms involved, we developed a model of radiation-induced senescence in cultured cancer cells, which exhibited a typical senescent phenotype, including upregulation of p53 and its target p21, followed by sustained reduction in cellular proliferation, changes in cell size and cytoskeleton organization, and senescence-associated beta-galactosidase activity. A functional siRNA screen using a cell death-related library identified the mitochondrial Ser protease HtrA2 as necessary for senescence development. Mass spectrometry-based proteomic profiling of the senescent cells indicated downregulation of proteins involved in cell cycle progression and DNA repair, and upregulation of proteins associated with malignancy, while irradiation with HtrA2 inhibition upregulated cell proliferation components. In search of direct HtrA2 substrates following radiation, we determined that HtrA2 cleaves the intermediate filament protein vimentin, affecting its cytoplasmic organization. Ectopic expression of active cytosolic HtrA2 resulted in similar changes to vimentin filament assembly. Thus HtrA2, contributes to several hallmarks of senescence and is involved in the cytoskeletal reorganization that accompanies radiation-induced senescence. SummaryHere the authors identify the Ser protease HtrA2 as a novel mediator of radiation-induced senescence, necessary for sustained proliferation arrest and reorganization of the vimentin filament network.

cancer biology↗

bioRxiv2020

A nuclear role for ARGONAUTE-2 in regulation of neuronal alternative polyadenylation

Argonaute 2 (AGO2), the effector protein partner of microRNAs (miRNAs) in the cytoplasmic RNA induced silencing complex, is further involved in nuclear RNA processing. However, a role for AGO2 in regulation of alternative polyadenylation was not yet demonstrated. Here, we reveal unexpected abundance of AGO2 in mouse neuronal nuclei and characterize nuclear AGO2 interactors by mass spectrometry. We discover that AGO2 broadly regulated alternative polyadenylation (APA) in neuronal cells. Specifically, we demonstrate how two isoforms of Ret mRNA, which encodes a receptor tyrosine kinase are regulated by AGO2-depenent APA, affecting downstream GDNF signaling in primary motor neurons.

cell biology↗

bioRxiv2020

Bringing BOS to light: Uncovering the key enzyme in the biosynthesis of the neurotoxin β-ODAP in Grass Pea (Lathyrus sativus L.)

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in parts of Asia and Africa for food and forage. While being a highly nutritious and robust crop, able to survive both drought and floods, it produces a neurotoxic compound, {beta}-N-oxalyl-L-,{beta}-diaminopropionic acid ({beta}-ODAP), which can cause a severe neurological disorder if consumed as a main diet component. So far, the enzyme that catalyzes the formation of {beta}-ODAP has not been identified. By combining protein purification and enzymatic assays with transcriptomic and proteomic analyses, we were able to identify the enzyme {beta}-ODAP synthetase (BOS) from grass pea. We show that BOS is an HXXXD-type acyltransferase of the BAHD superfamily and that its crystal structure is highly similar to that of plant hydroxycinnamoyl transferases. The identification of BOS, more than 50 years after it was proposed, paves the way towards the generation of non-toxic grass pea cultivars safe for human and animal consumption.

plant biology↗

bioRxiv2020

ERBB2 drives YAP activation and EMT-like processes during cardiac regeneration

Cardiomyocyte (CM) loss after injury results in adverse remodelling and fibrosis, which inevitably lead to heart failure. ERBB2-Neuregulin and Hippo-YAP signaling pathways are key mediators of CM proliferation and regeneration, yet the crosstalk between these pathways is unclear. Here, we demonstrate in adult mice that transient over-expression (OE) of activated ERBB2 in CMs promotes cardiac regeneration in a heart failure model. OE CMs present an EMT-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration, and ECM turnover. Molecularly, we identified YAP as a critical mediator of ERBB2 signaling. In OE CMs, YAP interacts with nuclear envelope and cytoskeletal components, reflecting the altered mechanic state elicited by ERBB2. Hippo-independent activating phosphorylation on YAP at S352 and S274 were enriched in OE CMs, peaking during metaphase, and viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Taken together, we demonstrate a potent ERBB2-mediated YAP mechanosensory signaling, involving EMT-like characteristics, resulting in heart regeneration. HighlightsO_LIERBB2-driven regeneration of scarred hearts recapitulates core-EMT processes C_LIO_LIYAP is activated and required downstream to ERBB2 signaling in CMs C_LIO_LIYAP activity is mechanically driven by cytoskeleton and nuclear envelope remodeling C_LIO_LIYAP S274 and S352 phosphorylation is essential for CM mitosis C_LI

molecular biology↗