Biology subjects

Florea, B. I.

Publications and source records attributed to Florea, B. I..

5 recordsLinked to original sources

bioRxiv2026

Structural Basis of Substrate Selectivity and Catalysis in the Mycobacterial Long-Chain Acyl-CoA Carboxylase

Long-chain acyl-CoA carboxylase (LCC) is an essential enzyme complex in mycobacteria that generates acyl-CoA precursors for mycolic acid and complex lipid biosynthesis, yet its architecture and mechanism of substrate selection have remained unclear. Here we determine pre- and post-reaction states of the endogenous 868-kDa LCC complex from Mycobacterium smegmatis by cryo-electron microscopy at 2.1-3.7 [A] resolution. These structures visualize ATP-dependent redistribution of the biotin carboxyl carrier protein. LCC assembles into an asymmetric 8:2:4:2 organization of AccA3, AccD4, AccD5, and AccE5, with two biotin carboxylase modules flexibly tethered to a heterohexameric carboxyltransferase core. We define the structural basis of substrate selectivity within the CT core: AccD5 selectively binds the short-chain substrate C3-CoA, whereas AccD4 accommodates the long-chain substrate C16-CoA. In addition, we resolve AccD5-centered assemblies that associate with biotin carboxylase modules yet lack AccD4, providing structural evidence that distinct carboxyltransferase cores can engage shared modules to generate alternative holoenzyme architectures. Together, these findings define LCC and AccD5-centered assemblies as elements of a combinatorial acyl-CoA carboxylase platform and establish the structural principles governing assembly-specific function in mycobacteria.

biochemistry↗

bioRxiv2024

Using BONCAT To Dissect The Proteome Of S. aureus Persisters

Bacterial persisters are a subpopulation of cells that exhibit a transient non-susceptible phenotype in the presence of bactericidal antibiotic concentrations. This phenotype can lead to the survival and regrowth of bacteria after treatment, resulting in relapse of infections. As such, it is also a contributing factor to antibacterial resistance. Multiple processes are believed to cause persister formation, yet identifying the proteins expressed during the induction of the persister state has been difficult, because the persister-state is rare, transient and does not lead to genetic changes. In this study, we used Bio-Orthogonal Non-Canonical Amino Acid Tagging (BONCAT) to label, and retrieve, the proteome expressed during the persister state for different strains of methicillin-resistant Staphylococcus aureus. After incubating antibiotic-exposed bacteria with the methionine ortholog L-azidohomoalanine to label the proteins of persister cells, we retrieved labeled proteins using click chemistry-pulldown methodology. Analysis of the retrieved proteome fraction of Methicillin resistant Staphylococcus aureus (MRSA) and Vancomycin resistant Staphylococcus aureus (VRSA) under challenge with {beta}-lactam and fluoroquinolone antibiotics with Label Free Quantification - Liquid chromatography mass spectrometry (LFQ-LCMS) based proteomics reveals the upregulation of proteins involved in stringent response, cell wall biosynthesis, purine metabolism, ppGpp biosynthesis, two component systems (TCS), lipid metabolism, ABC transporters, D-alanine biosynthesis and L-proline degradation. Conversely, we observed a decline of proteins associated with amino acid biosynthesis and degradation, protein biosynthesis, protein modification, and carbohydrate metabolism, among others. These findings indicate that modification of translational activity in persister cells enables bacterial cells to induce an active defense to survive antibiotic pressure.

microbiology↗

bioRxiv2024

CellEKT: A robust chemical proteomics workflow to profile cellular target engagement of kinase inhibitors

The human genome encodes 518 protein kinases that are pivotal for drug discovery in various therapeutic areas such as cancer and autoimmune disorders. The majority of kinase inhibitors target the conserved ATP-binding pocket, making it difficult to develop selective inhibitors. To characterize and prioritize kinase-inhibiting drug candidates, efficient methods are desired to determine target engagement across the cellular kinome. In this study, we present CellEKT (Cellular Endogenous Kinase Targeting), an optimized and robust chemical proteomics platform for investigating cellular target engagement of endogenously expressed kinases using the sulfonyl fluoride-based probe XO44 and two new probes ALX005 and ALX011. The optimized workflow enabled the determination of the kinome interaction landscape of covalent and non-covalent drugs across over 300 kinases, expressed as half maximum inhibitory concentration (IC50), which were validated using distinct platforms like phosphoproteomics and NanoBRET. With CellEKT, target engagement profiles were linked to their substrate space. CellEKT has the ability to decrypt drug actions and to guide the discovery and development of drugs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/616061v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@5dd891org.highwire.dtl.DTLVardef@1353379org.highwire.dtl.DTLVardef@1c67382org.highwire.dtl.DTLVardef@1c964eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2019

Chemical genetics strategy to profile kinase target engagement reveals role of FES in neutrophil phagocytosis via SYK activation

Chemical tools and methods that report on target engagement of endogenously expressed protein kinases by small molecules in human cells are highly desirable. Here, we describe a chemical genetics strategy that allows the study of non-receptor tyrosine kinase FES, a promising therapeutic target for cancer and immune disorders. Precise gene editing was used in combination with a rationally designed, complementary fluorescent probe to visualize endogenous FES kinase in HL-60 cells. We replaced a single oxygen atom by a sulphur in a serine residue at the DFG-1 position of the ATP-binding pocket in an endogenously expressed kinase, thereby sensitizing the engineered protein towards covalent labeling and inactivation by a fluorescent probe. The temporal control offered by this strategy allows acute inactivation of FES activity both during myeloid differentiation and in terminally differentiated neutrophils. Our results show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis by activation of SYK kinase, a central regulator of immune function in neutrophils. This strategy holds promise as a target validation method for kinases.

physiology↗

bioRxiv2017

Qinichelins, novel catecholate-hydroxamate siderophores synthesized via a multiplexed convergent biosynthesis pathway

The explosive increase in genome sequencing and the advances in bioinformatic tools have revolutionized the rationale for natural product discovery from actinomycetes. In particular, this has revealed that actinomycete genomes contain numerous orphan gene clusters that have the potential to specify many yet unknown bioactive specialized metabolites, representing a huge unexploited pool of chemical diversity. Here, we describe the discovery of a novel group of catecholate-hydroxamate siderophores termed qinichelins (2-5) from Streptomyces sp. MBT76. Correlation between the metabolite levels and the protein expression profiles identified the biosynthetic gene cluster (BGC; named qch) most likely responsible for qinichelin biosynthesis. The structure of the molecules was elucidated by bioinformatics, mass spectrometry and NMR. Synthesis of the qinichelins requires the interplay between four gene clusters, for its synthesis and for precursor supply. This biosynthetic complexity provides new insights into the challenges scientists face when applying synthetic biology approaches for natural product discovery.\n\nPride repository reviewer account details:\n\nURL: https://www.ebi.ac.uk/pride/archive/login\n\nProject accession: PXD006577\n\nUsername: reviewer35793@ebi.ac.uk\n\nPassword: 3H0iM1FK

microbiology↗