Biology subjects

Marin, E.

Publications and source records attributed to Marin, E..

15 recordsLinked to original sources

bioRxiv2025

Structural basis for no retinal binding in flotillin-associated rhodopsins

Rhodopsins are light-sensitive membrane proteins capturing solar energy via a retinal cofactor covalently attached to a lysine residue. Several groups of rhodopsins were reported to lack the conserved lysine and showed no retinal binding. Recently, flotillin-associated rhodopsins (FArhodopsins) were identified and suggested to lack the typical retinal binding pocket despite preserving the lysine residue in many members of the group. Here we present cryo-EM structures of paralog FArhodopsin and proteorhodopsin from marine bacteria Pseudothioglobus. The structures revealed pentameric assemblies of both proteins similar to those of other microbial rhodopsins. We demonstrate no binding of retinal to the FArhodopsin despite preservation of the lysine residue and overall similarity of the protein fold and internal organization to those of the retinal-binding paralog. Mutational analysis confirmed that two amino acids, H84 and E120, prevent retinal binding within the FArhodopsin. Thus, our work provides insights into the natural retinal loss in microbial rhodopsins and might contribute to the further understanding of the FArhodopsin clade.

biophysics↗

bioRxiv2024

CryoRhodopsins: a comprehensive characterization of a new clade of microbial rhodopsins from cold environments

Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin group from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have dual functionality switching between inward transmembrane proton translocation and photosensory activity, both of which can be modulated with UV light. CryoR1 exhibits two subpopulations in the ground state, which upon light activation lead to transient photocurrents of opposing polarities. A distinguishing feature of the group is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by light, we demonstrate that the arginine stabilizes a UV-absorbing intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified group and demonstrate principles of the adaptation of these rhodopsins to low temperatures.

biophysics↗

bioRxiv2023

A novel subgroup of light-driven sodium pumps with an additional Schiff base counterion

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identified a new subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterized a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and showed that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a new subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

biophysics↗

bioRxiv2023

Asparagine availability controls germinal centre B cell homeostasis

Germinal centre (GC) B cells proliferate at some of the highest rates of any mammalian cell, yet the metabolic processes which enable this are poorly understood. We performed integrated metabolomic and transcriptomic profiling of GC B cells, and found that metabolism of the non-essential amino acid asparagine (Asn) was highly upregulated. Asn was conditionally essential to B cells, and its synthetic enzyme, asparagine synthetase (ASNS) was upregulated following their activation, particularly more markedly in the absence of Asn, through the integrated stress response sensor general control non-derepressible 2 (GCN2). When Asns is deleted B cell survival and proliferation in low Asn conditions were strongly impaired, and removal of environmental Asn by asparaginase or dietary restriction markedly compromised the GC reaction, impairing affinity maturation and the humoral response to influenza infection. Using stable isotope tracing and single cell RNA sequencing, we found that metabolic adaptation to the absence of Asn requires ASNS, and that oxidative phosphorylation, mitochondrial homeostasis, and synthesis of nucleotides was particularly sensitive to Asn deprivation. Altogether, we reveal that Asn metabolism acts as a key regulator of B cell function and GC homeostasis. The one sentence summaryAsparagine metabolism is a critical regulator of B cell function, maintaining the germinal centre reaction.

immunology↗

bioRxiv2022

LAT1 enables T cell activation under inflammatory conditions: a new therapeutic target for rheumatoid arthritis

ObjectiveTo assess the L-type amino acid transporter-1 (LAT1) as a possible therapeutic target for rheumatoid arthritis (RA). MethodsSynovial LAT1 expression was monitored by immunohistochemistry and transcriptomic datasets. The contribution of LAT1 to gene expression and immune synapse formation was assessed by RNA-sequencing and total internal reflection fluorescent (TIRF) microscopy, respectively. Mouse models of RA were used to assess the impact of therapeutic targeting of LAT1. ResultsLAT1 was strongly expressed by CD4+ T cells in the synovial membrane of patients with active RA and the level of expression correlated with levels of ESR and CRP as well as DAS-28 scores. Deletion of LAT1 in murine CD4+ T cells inhibited the development of experimental arthritis and prevented the differentiation of CD4+ T cells expressing IFN-{gamma} and TNF-, without affecting regulatory T cells. LAT1 deficient CD4+ T cells demonstrated reduced transcription of genes associated with TCR/CD28 signalling, including Akt1, Akt2, Nfatc2, Nfkb1 and Nfkb2. Functional studies using TIRF microscopy revealed a significant impairment of immune synapse formation with reduced recruitment of CD3{zeta} and phospho-tyrosine signalling molecules in LAT1 deficient CD4+ T cells from the inflamed joints but not the draining lymph nodes of arthritic mice. Finally, it was shown that a small molecule LAT1 inhibitor, currently undergoing clinical trials in man, was highly effective in treating experimental arthritis in mice. ConclusionsIt was concluded that LAT1 plays a critical role in activation of pathogenic T cell subsets under inflammatory conditions and represents a promising new therapeutic target for RA. Key MessagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LILAT1 is an amino acid transporter that has previously been shown to play a role in T cell activation. C_LI What does this study add?O_LILAT1 is expressed by synovial T cells in human rheumatoid arthritis and the level of expression correlates with disease severity. C_LIO_LILAT1 expression by T cells is necessary for development of severe arthritis in animal models. C_LIO_LILAT1 is required for immune synapse formation and activation of pathogenic CD4+ T cell subsets in the inflamed joint, but not the lymph nodes. C_LIO_LIA small molecular weight LAT1 inhibitor, currently in clinical trials for cancer, is highly effective in animal models of rheumatoid arthritis. C_LI How might this impact on clinical practice of future developments?O_LIThe context-specific nature of LAT1 involvement in T cell activation positions it as an ideal therapeutic target to distinguish between pathogenic and protective T cell responses and this study provides the scientific rationale for clinical evaluation of LAT1 inhibitors in the treatment of rheumatoid arthritis. C_LI

immunology↗

bioRxiv2022

Structural insights into 3Fe-4S ferredoxins diversity in M.tuberculosis highlighted by a first redox complex with P450.

Ferredoxins are small iron-sulfur proteins and key players in essential metabolic pathways. Among all types, 3Fe-4S ferredoxins are less studied mostly due to anaerobic requirements. Their complexes with cytochrome P450 redox partners have not been structurally characterized. In the present work, we solved the structures of both 3Fe-4S ferredoxins from M. tuberculosis - Fdx alone and the fusion FdxE-CYP143. Our SPR analysis demonstrated a high affinity binding of FdxE to CYP143. According to SAXS data, the same complex is present in solution. The structure reveals extended multipoint interactions and the shape/charge complementarity of redox partners. Furthermore, FdxE binding induced conformational changes in CYP143 as evident from the solved CYP143 structure alone. The comparison of FdxE-CYP143 and modeled Fdx-CYP51 complexes further revealed the specificity of ferredoxins. Our results illuminate the diversity of electron transfer complexes for the production of different secondary metabolites.

biochemistry↗

bioRxiv2022

Structural basis for receptor selectivity and inverse agonism in S1P5 receptors

The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PR) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 [A] resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric subpocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, the new S1P5-inverse agonist structure sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR-family.

biophysics↗

bioRxiv2021

Antibacterial 3D-printed PMMA/ceramic composites

Following the rising interested on 3D-printed technologies, this research explores the possibility to use stereo-lithography to 3D print PMMA resins reinforced with up to 15% in weight of antibacterial ceramic powders. Three different reinforcements were tested, following previous literature data: aluminum nitride, titanium oxide and barium titanate. Between the three powders, the most uniform dispersion was achieved using aluminum nitride. Initial screenings with mixed and cured composite resins showed that between the three composite materials, only aluminum nitride or barium titanate PMMA showed a clear antibacterial effect when compared to the pristine reference, with aluminum nitride being the most effective against E. coli. When 3D printed using stereo-lithography, the composite containing aluminum nitride showed an even higher degree of dispersion and comparable antibacterial effects. Moreover, aluminum nitride reinforced PMMA resins showed good mechanical properties, comparable to the basic resin, and could be further strengthened by a standard post-curing process.

bioengineering↗

bioRxiv2021

Characterization of IL-22BP expression during Crohn's disease shows lack of evidence for IL-22BP production by T cells

Crohns disease (CD), a form of inflammatory bowel disease (IBD), is characterized by impaired epithelial barrier functions and dysregulated mucosal immune responses. IL-22 binding protein (IL-22BP) is a soluble inhibitor regulating IL-22 bioactivity, a cytokine proposed to play protective roles during CD. We and others have shown that IL-22BP is produced in IBD inflamed tissues, hence suggesting a role in CD. In this work, we extended the characterization of IL-22BP production and distribution in CD tissues by applying enzyme-linked immunosorbent assays to supernatants obtained from the culture of endoscopic biopsies of patients, and reverse transcription-quantitative polymerase chain reaction on sorted immune cell subsets. We reveal that IL-22BP levels are higher in inflamed ileums than colons. We observe that in a cell-intrinsic fashion, populations of mononuclear phagocytes and eosinophils express IL-22BP at the highest levels in comparison to other sources of T cells. We suggest the enrichment of intestinal eosinophils could explain higher IL-22BP levels in the ileum. In inflamed colon, we reveal the presence of increased IL-22/IL22BP ratios compared to controls, and a strong correlation between IL-22BP and CCL24. We identify monocyte-derived dendritic cells (moDC) as a cellular subtype co-expressing both cytokines and validate our finding using in vitro culture systems. We also show that retinoic acid induces the secretion of both IL-22BP and CCL24 by moDC. Finally, we report on higher IL-22BP levels in active smokers. In conclusion, our work provides new information relevant to therapeutic strategies modulating IL-22 bioactivity in CD, especially in the context of disease location.

immunology↗

bioRxiv2020

A new twist of rubredoxin function in M. tuberculosis

Electron transfer mediated by metalloproteins drives many biological processes. Rubredoxins are ubiquitous iron-containing electron carriers that play important roles in bacterial adaptation to changing environmental conditions. In Mycobacterium tuberculosis, oxidative and acidic stresses as well as iron starvation induce rubredoxin expression. However, their functions during M. tuberculosis infection is unknown. In the present work, we show that rubredoxin B (RubB) supports catalytic activity of mycobacterial cytochrome P450s, CYP124, CYP125, and CYP142, which are important for bacterial viability and pathogenicity. We solved the crystal structure of RubB and characterized the interaction between RubB and CYPs using site-directed mutagenesis. Mutations that neutralized single charge on the surface of RubB did not dramatically decrease activity of studied CYPs, and isothermal calorimetry (ITC) experiments indicated that interactions are transient and not highly specific. Our findings suggest that a switch from ferredoxins to rubredoxins support CYP activity in M. tuberculosis-infected macrophages. Our electrochemical experiments suggest potential applications of RubB in biotechnology.

biochemistry↗

bioRxiv2020

The connectome of the adult Drosophila mushroom body: implications for function

Making inferences about the computations performed by neuronal circuits from synapse-level connectivity maps is an emerging opportunity in neuroscience. The mushroom body (MB) is well positioned for developing and testing such an approach due to its conserved neuronal architecture, recently completed dense connectome, and extensive prior experimental studies of its roles in learning, memory and activity regulation. Here we identify new components of the MB circuit in Drosophila, including extensive visual input and MB output neurons (MBONs) with direct connections to descending neurons. We find unexpected structure in sensory inputs, in the transfer of information about different sensory modalities to MBONs, and in the modulation of that transfer by dopaminergic neurons (DANs). We provide insights into the circuitry used to integrate MB outputs, connectivity between the MB and the central complex and inputs to DANs, including feedback from MBONs. Our results provide a foundation for further theoretical and experimental work.

neuroscience↗

bioRxiv2020

Hydroxylation of antitubercular drug candidate, SQ109, by mycobacterial cytochrome P450

Spreading of the multidrug-resistant (MDR) strains of the deadliest pathogen Mycobacterium tuberculosis (Mtb) generates the need for new effective drugs. SQ109 showed activity against resistant Mtb and already advanced to Phase II/III clinical trials. Fast SQ109 degradation is attributed to the human liver Cytochrome P450s (CYPs). However, no information is available about interactions of the drug with Mtb CYPs. Here, we show that Mtb CYP124, previously assigned as a methyl-branched lipid monooxygenase, binds and hydroxylates SQ109 in vitro. A 1.25 [A]-resolution crystal structure of the CYP124-SQ109 complex unambiguously shows two conformations of the drug, both positioned for hydroxylation of the {omega}-methyl group in the trans position. The hydroxylated SQ109 presumably forms stabilizing H-bonds with its target, the Mycobacterial membrane protein Large 3 (MmpL3). We anticipate that Mtb CYPs could function as analogs of drug-metabolizing human CYPs affecting pharmacokinetics and pharmacodynamics of antitubercular (anti-TB) drugs.

biochemistry↗

bioRxiv2020

Metabolic fate of human immunoactive sterols in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266, the most potent among them, can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system.Competing Interest StatementA.Gi. and E.S. are employees of MT-Medicals LLC. The other authors declare no competing interests.View Full Text

biochemistry↗

bioRxiv2020

Rapid Inactivation of SARS-CoV-2 by Silicon Nitride, Copper, and Aluminum Nitride

IntroductionViral disease spread by contaminated commonly touched surfaces is a global concern. Silicon nitride, an industrial ceramic that is also used as an implant in spine surgery, has known antibacterial activity. The mechanism of antibacterial action relates to the hydrolytic release of surface disinfectants. It is hypothesized that silicon nitride can also inactivate the coronavirus SARS-CoV-2. MethodsSARS-CoV-2 virions were exposed to 15 wt.% aqueous suspensions of silicon nitride, aluminum nitride, and copper particles. The virus was titrated by the TCD50 method using VeroE6/TMPRSS2 cells, while viral RNA was evaluated by real-time RT-PCR. Immunostaining and Raman spectroscopy were used as additional probes to investigate the cellular responses to virions exposed to the respective materials. ResultsAll three tested materials showed >99% viral inactivation at one and ten minutes of exposure. Degradation of viral RNA was also observed with all materials. Immunofluorescence testing showed that silicon nitride-treated virus failed to infect VeroE6/TMPRSS2 cells without damaging them. In contrast, the copper-treated virus suspension severely damaged the cells due to copper ion toxicity. Raman spectroscopy indicated differential biochemical cellular changes due to infection and metal toxicity for two of the three materials tested. ConclusionsSilicon nitride successfully inactivated the SARS-CoV-2 in this study. The mechanism of action was the hydrolysis-mediated surface release of nitrogen-containing disinfectants. Both aluminum nitride and copper were also effective in the inactivation of the virus. However, while the former compound affected the cells, the latter compound had a cytopathic effect. Further studies are needed to validate these findings and investigate whether silicon nitride can be incorporated into personal protective equipment and commonly touched surfaces, as a strategy to discourage viral persistence and disease spread.

microbiology↗

bioRxiv2020

Molecular mechanism of light-driven sodium pumping

Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience. An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters. KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies. The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release. Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics.

biophysics↗