Biology subjects

Kuzmin, A.

Publications and source records attributed to Kuzmin, A..

11 recordsLinked to original sources

bioRxiv2026

A PSMA-Targeted Up-conversion Nanoplatform for Deep-Tissue dual activation Photodynamic and Sonodynamic Therapy of Castration Resistant Prostate Cancer.

Photodynamic therapy (PDT) for prostate cancer is limited by the shallow penetration of visible light into deep-seated tumors. In contrast, sonodynamic therapy (SDT) enables noninvasive deep-tissue activation using ultrasound, while near-infrared photodynamic therapy (NIR-PDT) enhances tissue penetration through up-conversion-mediated activation. Based on these advantages, we developed a prostate-specific membrane antigen (PSMA)-targeted nanotheranostic platform (UCNPs@mSiO2/HPPH@TCS) to compare NIR-PDT and SDT in vitro for penetration-enhanced and targeted prostate cancer therapy. The nanoformulation consists of NaYF4:Yb3+, Er3+ up-conversion nanoparticles coated with mesoporous silica for high loading of HPPH (Photochlor), which functions as both photosensitizer and sonosensitizer, and a PSMA-targeted chitosan shell for selective tumor targeting. Physicochemical characterization confirms a uniform core-shell structure ([~]63 nm). Tissue-mimicking depth studies demonstrate that SDT and NIR-PDT achieve greater penetration than conventional 665 nm PDT in thicker tissue layers. Intracellular reactive oxygen species (ROS) analysis shows that SDT generates higher total ROS levels, whereas NIR-PDT produces greater singlet oxygen generation. Three-dimensional spheroid models further validate efficacy, demonstrating rapid spheroid collapse via apoptosis, with SDT inducing earlier apoptosis and more uniform penetration than PDT. Collectively, the results suggest SDT provides superior overall therapeutic efficacy compared with NIR-PDT, highlighting the potential of this nanoplatform for precision prostate cancer therapy.

cancer biology↗

bioRxiv2025

Oxidative Stress, Hypoxia and Cellular Metabolism: Unraveling the Effects of Fentanyl on Lung Cancer Cells

Fentanyl, a widely used opioid analgesic for cancer pain management, is effective but requires cautious administration due to its potential for respiratory depression. Beyond its analgesic properties, fentanyls broader impact on cancer biology and biochemical alterations in lung carcinoma cells remains underexplored. This study investigates fentanyls influence on oxidative stress, mitochondrial function, hypoxia-inducing factors, apoptosis, and cytokine production in A549 lung cancer cells. Our findings reveal that fentanyl increases reactive oxygen species (ROS) generation, disrupts cellular homeostasis, induces DNA damage, and alters key signaling pathways, potentially affecting tumor metabolism and progression. Our "Ramanomics" data further highlight fentanyl-driven changes in key ions (inorganic phosphate, calcium) and biomolecules (glycogen, phospholipids, proteins, nucleic acids, and enzymes) at subcellular mitochondrial levels. These insights contribute to understanding fentanyls mechanistic impact on lung cancer progression and may inform optimized therapeutic strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/665412v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1fb7aaaorg.highwire.dtl.DTLVardef@13255deorg.highwire.dtl.DTLVardef@1a398cdorg.highwire.dtl.DTLVardef@184d63e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Discovery of excited state proton transfer in flavin-based fluorescent protein with large Stokes shift

Flavin-binding proteins (flavoproteins) are widespread in nature, revealing versatile oxidation-reduction reactions and photochemistry. Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics, as well as fluorescent markers and photogenerators of reactive oxygen species. Despite extensiev efforts, all currently used LOV-derived proteins have similar absorption spectra with maxima around 275, 35-0375, and 450-485 nm. Here, we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein, LSSFbFP, which can be obtainedin vivo and in vitro, with absorption maxima at 340-350 and 395-405 nm. Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ~500 nm. We sho that the protein binds lumichrome as the chromophore and use low temperature and time-resolved spectroscopy, X-ray crystallography and modeling to prove that the apparent Stokes shift of LSSFbFP occurs due to excited state proton phenomena observed in flavoproteni s and pave the way for engineering of new flavin-based molecular instruments.

biochemistry↗

bioRxiv2025

ER-Localized Ceramide Accumulation Due to Disrupted CERT Transport Contributes to Replicative Senescence

Ceramides regulate diverse cellular processes through compartment-specific accumulation. While mitochondrial ceramide accumulation promotes apoptosis, its regulation and function during senescence remain incompletely understood. Here, we integrate lipidomics, transcriptomics, Raman spectroscopy and biochemical characterizations to define sphingolipid remodeling in replicative senescence. Senescent cells exhibit elevated ceramide levels and depletion of very-long-chain sphingomyelins, despite unaltered sphingomyelin synthase 1 expression, implicating impaired ceramide-sphingomyelin turnover. Pharmacological inhibition of ceramide transfer protein (CERT), the ER-to-Golgi ceramide transporter, phenocopies this sphingolipid remodeling and enhances senescence markers, suggesting disrupted ceramide trafficking as a driver of senescence. Raman spectroscopy suggests ceramide accumulation localized to the ER, with no enrichment at mitochondria, consistent with a non-apoptotic phenotype. Analysis of ER-enriched fractions confirm increased ceramide levels in ER fractions of senescent cells. Mechanistically, ceramide accumulation at the ER can contribute to ER stress, a key component of the senescence program. These findings identify altered ceramide trafficking as a contributor to ER stress and highlight ER-localized ceramide as a critical component of senescence-associated sphingolipid remodeling. SignificanceHow ceramides function during replicative senescence has remained poorly understood. In this study, we demonstrate that senescent cells accumulate ceramides at the endoplasmic reticulum (ER), revealing a fundamentally different mode of ceramide regulation and function in senescence. By integrating lipidomics, enzymatic assays, Raman-BCA imaging, and biochemical assays, we show that impaired ER-to-Golgi ceramide transport leads to ER-localized ceramide retention. Disrupting ceramide trafficking through inhibition of the ceramide transfer protein CERT phenocopies senescence-associated lipid remodeling and enhances senescence markers, establishing altered ceramide transport as a mechanistic contributor to this process. These findings link impaired ceramide trafficking to replicative senescence and suggest that ceramide function in senescence is linked to its retention at the ER.

cell biology↗

bioRxiv2024

Engineering of soluble bacteriorhodopsin

Bacteriorhodopsin is a seven-helical light-driven proton pump and a model membrane protein. Here, we report engineering of soluble analogues of bacteriorhodopsin, NeuroBRs, which bind retinal and photocycle under illumination. We also report the crystallographic structure of NeuroBR_A, determined at anisotropic resolution reaching 1.76 [A], that reveals a conserved chromophore binding pocket and tertiary structure. Our results highlight the power of modern protein engineering approaches and pave the way towards wider development of molecular tools derived from membrane proteins.

bioengineering↗

bioRxiv2024

High-throughput algorithm predicts F-Type ATP synthase rotor ring stoichiometries of 8 to 27 protomers

ATP synthases are large enzymes present in every living cell. They consist of a transmembrane and a soluble domain, each comprising multiple subunits. The transmembrane part contains an oligomeric rotor ring (c-ring), whose stoichiometry defines the ratio between the number of synthesized ATP molecules and the number of ions transported through the membrane. Currently, c-rings of F-Type ATP synthases consisting of 8 to 17 (except 16) subunits have been experimentally demonstrated. Here, we present an easy-to-use high-throughput computational approach based on AlphaFold that allows us to estimate the stoichiometry of all homooligomeric c-rings, whose sequences are present in genomic databases. We validate the approach on the available experimental data, obtaining the correlation as high as 0.94 for the reference data set, and use it to predict the existence of c-rings with stoichiometry varying from 8 to 27. We then conduct molecular dynamics simulations of two c-rings with stoichiometry above 17 to corroborate the machine learning-based predictions. Our work strongly suggests existence of rotor rings with previously undescribed high stoichiometry in natural organisms and highlights the utility of AlphaFold-based approaches for studying homooligomeric proteins.

bioinformatics↗

bioRxiv2023

Reengineering of a flavin-binding fluorescent protein using ProteinMPNN

Recent advances in machine learning techniques have led to development of a number of protein design and engineering approaches. One of them, ProteinMPNN, predicts an amino acid sequence that would fold and match user-defined backbone structure. In this short report, we test whether ProteinMPNN can be used to reengineer a flavin-binding fluorescent protein, CagFbFP. We fixed the native backbone conformation and the identity of 20 amino acids interacting with the chromophore (flavin mononucleotide, FMN), while letting ProteinMPNN predict the rest of the sequence. The software package suggested replacing 36-48 out of the remaining 86 amino acids. The three designs that we tested experimentally displayed different expression levels, yet all were able to bind FMN and displayed fluorescence, thermal stability and other properties similar to those of CagFbFP. Our results demonstrate that ProteinMPNN can be used to generate diverging unnatural variants of fluorescent proteins, and, more generally, to reengineer proteins without losing their ligand-binding capabilities.

bioengineering↗

bioRxiv2023

Two distinct mechanisms of flavoprotein spectral tuning revealed by low-temperature and time-dependent spectroscopy

Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time-resolved and low temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low-temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine X-ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color tuning mechanisms of flavin-binding proteins and pave the way for engineering of new variants with improved optical properties. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/549366v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1403d8eorg.highwire.dtl.DTLVardef@894bd1org.highwire.dtl.DTLVardef@1d089b1org.highwire.dtl.DTLVardef@1ffd3c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Structure and dynamics of the SARS-CoV-2 envelope protein monomer

Coronaviruses, especially SARS-CoV-2, present an ongoing threat for human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic -helical transmembrane domain (TMD) and amphiphilic -helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43 and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane. This may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate budding of the viral particles. The presented results may be helpful for better understanding of the function of coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.

biophysics↗

bioRxiv2020

IDH1 Mutations Induce Organelle Defects Via Dysregulated Phospholipids

Cytosolic IDH1 enzyme plays a key, but currently unexplored, role in lipid biosynthesis. Using Raman imaging microscopy, we identified heterogeneous lipid profiles in cellular organelles attributed uniquely to IDH1 mutations. Via organelle lipidomics, we found an increase in saturated and monounsaturated fatty acids in the endoplasmic reticulum of IDH1mut cells compared with IDHWT glioma. We showed that these fatty acids incorporate into phospholipids and induce organelle dysfunctions, with prominent dilation of Golgi apparatus, which can be restored by transient knockdown of stearyl-CoA desaturase or inhibition of D-2-hydroxyglutarate (D-2HG) formation. We validated these findings using tissue from patients with glioma. Oleic acid addition led to increased sensitivity to apoptosis of IDH1mut cells compared with IDHWT. Addition of D-2HG to U251WT cells lead in increased ER and Golgi apparatus dilation. Collectively, these studies provide clinically relevant insights into the functional link between IDH1mut-induced lipid alterations and organelle dysfunction, with therapeutic implications. SignificanceGliomas are devastating tumors, with the most aggressive form--glioblastoma multiforme-- correlated with a mean patient survival of 14.5 months. No curative treatment exists to date. Low-grade glioma (LGG) with the isocitrate dehydrogenase 1 (IDH1) mutation, R132H, provides a survival benefit to patients. Understanding the unique metabolic profile of IDH1mut could provide clues regarding its association with longer survival and information about therapeutic targets. Herein, we identified lipid imbalances in organelles, generated by IDHmut in cells and patient tissue, that were responsible for Golgi dilation and that correlated with increased survival. Addition of oleic acid, which tilted the balance towards elevated levels of monounsaturated fatty acids produced IDH1mut-specific cellular apoptosis. HighlightsO_LISingle-organelle omics revealed unique alterations in lipid metabolism due to IDH1-mutations. C_LIO_LIIDH mutation leads to organelle-wide structural defects. C_LIO_LIIDH1 mutation leads to increased monounsaturated fatty acids levels in glioma cells and oligodendroglioma patient samples. C_LIO_LILipid alterations affect the membrane integrity of the Golgi apparatus. C_LIO_LIIncreased D-2HG induced SCD expression and elevated monounsaturated fatty acids C_LIO_LITilting the balance toward more-abundant monounsaturated fatty acids leads to specific IDH1mut glioma apoptosis. C_LI

cancer biology↗

bioRxiv2019

The majority of histone acetylation is a consequence of transcription

Histone acetylation is a ubiquitous hallmark of transcriptional activity, but whether the link is of a causal or consequential nature is still a matter of debate. In this study we resolve this question. Using both immunoblot analysis and chromatin immunoprecipitation-sequencing (ChIP-seq) in S. cerevisiae, we show that the majority of histone acetylation is dependent on transcription. Loss of histone H4 acetylation upon transcription inhibition is partially explained by depletion of histone acetyltransferases (HATs) from gene bodies, implicating transcription in HAT targeting. Despite this, HAT occupancy alone poorly predicts histone acetylation, suggesting that HAT activity is regulated at a step post-recruitment. Collectively, these data show that the majority of histone acetylation is a consequence of RNAPII promoting both the recruitment and activity of histone acetyltransferases.

molecular biology↗