Biology subjects

Bashiri, G.

Publications and source records attributed to Bashiri, G..

11 recordsLinked to original sources

bioRxiv2026

Tumor-associated mammary fibroblast matrices contain O-glycosylated oncofetal fibronectin

Oncofetal fibronectin (oFN) is a known biomarker of cancer progression and is involved in events such as epithelial mesenchymal transition and multidrug resistance. It is also understood that fibronectin (FN) and its splice variants are differentially arranged by cancer associated fibroblasts (CAFs) to support cancer cell proliferation and migration. Contrastingly, little is understood about the mechanobiology of oFN, a post-translationally modified FN isoform defined by O-glycosylation of a specific threonine residue. To investigate the architectural features of the oFN matrix, CAFs were simulated by culturing human mammary fibroblasts (HMFs) on stiff polyacrylamide gels (20 kPa) in media collected from HCC-1937 (primary site breast adenocarcinoma) and MDA-MB-231 (metastatic breast adenocarcinoma) cells. As a control, HMFs were also cultured on softer gels representing healthy adipose tissue (2 kPa) in media not exposed to cancer cells. After immunocytochemical analysis, tumor conditioned HMFs displayed an expected CAF-like phenotype that was most prominent with MDA-MB-231 conditioned media. Matrices produced by tumor-conditioned HMFs contained more oFN with structural features mostly following that of the overall dense, aligned FN matrix. Cancer cells reseeded on their corresponding decellularized tumor-associated matrices had greater F-actin/oFN colocalization than control matrix. The lack of distinct oFN topographical cues may indicate that oFN O-glycosylation is more directly involved in biochemical signaling than mechanosignaling through matrix architecture. Together, this study established a platform for investigating stromal oFN which revealed that tumor-conditioned media sourced from cancer cells with different metastatic potential influences HMF activation, leading to distinct total FN and thus oFN matrix microarchitecture features.

bioengineering↗

bioRxiv2026

Tumor-Associated EDA-FN-Enriched Matrix Instructs Macrophage Behavior

IntroductionCancer progression is driven not only by tumor cells but also by interactions between the extracellular matrix (ECM), stromal cells, and immune cells within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are major drivers of ECM remodeling, assembling ECM with aberrant organization. Extra domain A fibronectin (EDA-FN), a cellular FN containing an extra type III domain, is upregulated in the TME. EDA-FN regulates cellular behavior and has been associated with poor patient prognosis. Macrophages are among the most abundant immune cells within the TME, where they contribute to TME remodeling and inflammation to promote cancer cell invasion and metastasis. However, how tumor-associated matrix-specific cues regulate macrophage behavior remains largely understudied. PurposeHere, we developed a fibroblast-derived matrix platform that captures the structural imprint of tumor-associated EDA-enriched matrices and investigated how matrix-specific cues regulate macrophage behavior in the absence of ongoing soluble factor cues. MethodHuman mammary fibroblasts (HMFs) preconditioned in incubated low-serum media (lNC, or control) and MDA-MB231 metastatic breast cancer cell-conditioned media (mTCM) were cultured on polyacrylamide gels of 2 kPa and 20 kPa, respectively, followed by decellularization. Matrix organization, including fiber alignment, width, and intrafibrillar spacing, was quantified from confocal images. Decellularized EDA-FN-enriched matrices were subsequently reseeded with macrophages to assess macrophage morphology, phenotype, and matrix interactions. ResultsThe combined effects of tumor-derived soluble factors and pathological stiffness induced a CAF-like phenotype in HMFs, accompanied by cytoskeletal reorganization and microarchitectural alterations of EDA-FN-enriched matrices. Tumor-associated matrices exhibited increased alignment, narrower fiber width, and enlarged intrafibrillar spacing compared to control matrices. These aberrant, tumor-associated matrix-derived features were associated with altered macrophage behavior, including heterogeneous morphology, enhanced localized EDA-FN matrix loss beneath the cell body, and a hybrid phenotype with a shift toward a CD206-dominant profile. ConclusionsThese findings demonstrate the feasibility of obtaining EDA-FN-enriched matrices to isolate matrix-specific cues for investigating macrophage-ECM interactions. Furthermore, this platform can be leveraged to identify matrix-targeting therapeutic approaches for modulating macrophage function within the TME.

bioengineering↗

bioRxiv2026

Time-resolved cryo-EM reveals conformational trajectory of allosteric activation in isocitrate lyase

Isocitrate lyase 2 (ICL2) from Mycobacterium tuberculosis undergoes dramatic conformational rearrangements upon binding to the allosteric effector acetyl-CoA. Time-resolved cryo-EM captured conformational states along the ICL2 activation trajectory, revealing how acetyl-CoA binding at the allosteric sites leads to asymmetric, half-of-site activity at the catalytic centres. These findings support a conformational selection model of allostery, whereby acetyl-CoA binding shifts the pre-existing equilibrium towards an active state of the enzyme.

biochemistry↗

bioRxiv2025

Dynamic allostery drives acetyl-CoA-mediated activation of Mycobacterium tuberculosis isocitrate lyase 2

Mycobacterium tuberculosis isocitrate lyase 2 (ICL2) is an allosterically regulated enzyme that enables the bacterium to survive on non-glycolytic substrates during infection. Previous studies showed that ICL2 is allosterically regulated by acetyl-CoA and its analogues but the molecular mechanism underpinning this regulation is unknown. Here, we use protein NMR, crystallography, molecular dynamics, and mutagenesis studies to show that two unique structural features of ICL2, its C-terminal domain and a unique helical substructure on its N-terminal catalytic domain, play important roles in the enzymes allostery. In particular, we found that the binding of acetyl-CoA promotes the dimerisation of the C-terminal domain and disrupts its interactions with the unique helical substructure on the N-terminal domain. This leads to conformational changes in the ICL2 enzyme that induces activation. Taken together, our findings reveal, for the first time, how the binding of acetyl-CoA, which is not an ICL2 substrate, induces ICL2 activation. By extension, the work also identifies a novel allosteric mechanism controlling M. tuberculosis metabolism that is amenable to therapeutic manipulation. Significance StatementMycobacterium tuberculosis isocitrate lyase 2 (ICL2) was previously shown to be activated by acetyl-CoA and propionyl-CoA - two central metabolites generated by the metabolism of sugars and fatty acids. However, it is not known how the binding of these metabolites leads to the activation of ICL2. Together with its isoform ICL1, ICL2 has been shown to be essential for the survival and pathogenesis of the bacterium. Understanding how this regulation occurs can help design novel treatments to target this protein and eradicate these bacteria, which cause the most deaths worldwide due to a single bacterial agent. This system also presents a fascinating model to examine allostery in proteins, with the techniques illustrated in this paper being applicable to other allosteric proteins.

biochemistry↗

bioRxiv2025

Feedback regulation of iron-sulfur cluster biogenesis

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in biological systems. Given their central role in bacterial metabolism and pathogenesis, the biogenesis of Fe-S clusters is tightly controlled. We reveal a feedback regulatory mechanism involving the sulfide producing SufS/SufU complex within the sulfur utilization (SUF) system of Mycobacterium tuberculosis, the bacterium that causes tuberculosis. In this mechanism, [2Fe-2S] clusters compete with zinc ions for binding to the sulfide transfer protein SufU. Cluster binding induces SufU tetramerization, which prevents its interaction with the cysteine desulfurase SufS, thereby inhibiting SufS activation and limiting sulfide supply for Fe-S cluster biogenesis. These findings uncover an unrecognized regulatory mechanism in M. tuberculosis, ensuring strict control of Fe-S cluster production.

biochemistry↗

bioRxiv2024

EDA Fibronectin Microarchitecture and YAP Translocation During Wound Closure

Fibronectin (Fn) is an extracellular matrix glycoprotein with mechanosensitive structure-function. EDA Fn, a Fn isoform, is not present in adult tissue but is required for tissue repair. Curiously, EDA Fn is linked to both regenerative and fibrotic tissue repair. Given that Fn mechanoregulates cell behavior, Fn EDA organization during wound closure might play a role in mediating these differing responses. One mechanism by which cells sense and respond to their microenvironment is by activating a transcriptional co-activator, Yes-associated protein (YAP). Interestingly, YAP activity is not only required for wound closure, but similarly linked to both regenerative and fibrotic repair. Therefore, this study aims to evaluate how, during normal and fibrotic wound closure, EDA Fn organization might modulate YAP translocation by culturing human dermal fibroblasts on polydimethylsiloxane (PDMS) substrates mimicking normal (soft: 18 kPa) and fibrotic (stiff: 146 kPa) wounded skin. On stiffer substrates mimicking fibrotic wounds, fibroblasts assembled an aligned EDA Fn matrix comprising thinner fibers, suggesting increased microenvironmental tension. To evaluate if cell binding to the EDA domain of Fn was essential to overall matrix organization, fibroblasts were treated with Irigenin, which inhibits binding to the EDA domain within Fn. Blocking adhesion to EDA led to randomly organized EDA Fn matrices with thicker fibers, suggesting reduced microenvironmental tension even during fibrotic wound closure. To evaluate if YAP signaling plays a role in EDA Fn organization, fibroblasts were treated with CA3, which suppresses YAP activity in a dose-dependent manner. Treatment with CA3 also led to randomly organized EDA Fn matrices with thicker fibers, suggesting a potential connected mechanism of reducing tension during fibrotic wound closure. Next, YAP activity was assessed to evaluate the impact of EDA Fn organization. Interestingly, fibroblasts migrating on softer substrates mimicking normal wounds increased YAP activity but on stiffer substrates, decreased YAP activity. When fibroblasts on stiffer substrates were treated with Irigenin or CA3, fibroblasts increased YAP activity. These results suggest there may be disrupted signaling between EDA Fn organization and YAP translocation during fibrotic wound closure that could be restored when reestablishing normal EDA Fn matrix organization to instead drive regenerative wound repair.

bioengineering↗

bioRxiv2022

Allosteric inhibition of Staphylococcus aureus MenD by 1,4-dihydroxy naphthoic acid: A feedback inhibition mechanism of the menaquinone biosynthesis pathway

Menaquinones (MKs) are electron carriers in bacterial respiratory chains. In Staphylococcus aureus (Sau), MKs are essential for aerobic and anaerobic respiration. As MKs are redox-active, their biosynthesis likely requires tight regulation to prevent disruption of cellular redox balance. We recently found that the Mycobacterium tuberculosis MenD, the first committed enzyme of the MK biosynthesis pathway, is allosterically inhibited by the downstream metabolite 1,4-dihydroxy-2-naphthoic acid (DHNA). To understand if this is a conserved mechanism in phylogenetically distant genera that also utilize MK, we investigated whether the Sau-MenD is allosterically inhibited by DHNA. Our results show that DHNA binds to and inhibits SEPHCHC synthase activity of Sau-MenD enzymes. We identified residues in the DHNA binding pocket that are important for catalysis (Arg98, Lys283, Lys309) and inhibition (Arg98, Lys283). Furthermore, we show that exogenous DHNA inhibits growth of Sau, an effect that can be rescued by supplementing the growth media with MK-4. Our results demonstrate that despite a lack of strict conservation of the DHNA-binding pocket between Mtb-MenD and Sau-MenD, feedback inhibition by DHNA is a conserved mechanism in Sau-MenD and hence the Sau MK biosynthesis pathway. These findings may have implications for the development of anti-staphylococcal agents targeting MK biosynthesis

biochemistry↗

bioRxiv2020

Mutations in fbiD(Rv2983) as a novel determinant of resistance to pretomanid and delamanid in Mycobacterium tuberculosis

The nitroimidazole pro-drugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10-5 CFU. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 91% of which occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance: fbiC (56%), fbiA (15%), ddn (12%), fgd (4%) and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983, a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance, but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

microbiology↗

bioRxiv2019

Allosteric Regulation of Vitamin K2 Biosynthesis in a Human Pathogen

Menaquinone (Vitamin K2) plays a vital role in energy generation and environmental adaptation in many bacteria, including Mycobacterium tuberculosis (Mtb). Although menaquinone levels are known to be tightly linked to the redox/energy status of the cell, the regulatory mechanisms underpinning this phenomenon are unclear. The first committed step in menaquinone biosynthesis is catalyzed by MenD, a thiamine diphosphate-dependent enzyme comprising three domains. Domains I and III form the MenD active site, but no function has yet been ascribed to domain II. Here we show the last cytosolicmetabolite in the menaquinone biosynthesis pathway (1,4-dihydroxy-2-napthoic acid, DHNA) binds to domain II of Mtb-MenD and inhibits enzyme activity. We identified three arginine residues (Arg97, Arg277 and Arg303) that are important for both enzyme activity and the feedback inhibition by DHNA: Arg277 appears to be particularly important for signal propagation from the allosteric site to the active site. This is the first evidence of feedback regulation of the menaquinone biosynthesis pathway in bacteria, unravelling a protein level regulatory mechanism for control of menaquinone levels within the cell.

biochemistry↗

bioRxiv2018

A revised biosynthetic pathway for the cofactor F420 in bacteria

Cofactor F420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F420 has not been fully eluci-dated: neither the enzyme that generates the putative intermediate 2-phospho-L-lactate, nor the function of the FMN-binding C-terminal domain of the {gamma}-glutamyl ligase (FbiB) in bacteria are known. Here we show that the guanylyltransferases FbiD and CofC accept phosphoenolpyruvate, rather than 2-phospho-L-lactate, as their substrate, leading to the formation of the previously uncharacterized intermediate, dehydro-F420-0. The C-terminal domain of FbiB then utilizes FMNH2 to reduce dehydro-F420-0, which produces mature F420 species when combined with the {gamma}-glutamyl ligase activity of the N-terminal domain. This new insight has allowed the heterologous expression F420 from a recombinant F420 biosynthetic pathway in Escherichia coli.

molecular biology↗

bioRxiv2018

Mutations in Rv2983 as a novel determinant of resistance to nitroimidazole drugs in Mycobacterium tuberculosis

Delamanid represents one of two novel antimicrobial classes approved to treat tuberculosis in over 40 years. Pretomanid is another promising nitroimidazole pro-drug in clinical development. Characterization of the full spectrum of mutations conferring resistance to nitroimidazoles and their related phenotypes in Mycobacterium tuberculosis will inform development of suitable genotypic and phenotypic drug susceptibility tests. Here, we used a range of pretomanid doses to select pretomanid-resistant mutants in two pathologically distinct murine TB models. The frequency of spontaneous pretomanid resistance mutations was approximately 10-5 CFU. Pretomanid demonstrated dose-dependent bactericidal activity and selective amplification of resistant mutants. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 90% of which were found in 1 of 5 genes previously associated with nitroimidazole activation and resistance. The remaining 10% harbored isolated mutations in Rv2983. Complementing an Rv2983 mutant with a wild-type copy of Rv2983 restored wild-type susceptibility to pretomanid and delamanid, confirming that loss of Rv2983 function causes nitroimidazole resistance. By quantifying F420 and its precursor Fo in Mycobacterium smegmatis overexpressing Rv2983 and an M. tuberculosis Rv2983 mutant, we provide evidence that Rv2983 is necessary for F420 biosynthesis and nitroimidazole activation, perhaps as the guanylyltransferase CofC. F420H2-deficient mutants displayed hypersusceptibility to malachite green (MG), a selective decontaminant present in solid media used to isolate and propagate mycobacteria from clinical samples. The wide diversity of mutations causing high-level pretomanid resistance and MG hypersusceptibility of most mutants poses significant challenges to clinical detection of nitroimidazole resistance using either genotypic or phenotypic methods.\n\nSignificanceNitroimidazole pro-drugs represent a promising new class of anti-tuberculosis drugs. Reliable methods to assure nitroimidazole susceptibility are critical to assure their optimal use. Yet, the spectrum of nitroimidazole resistance mutations remains incompletely characterized. Using 161 pretomanid-resistant Mycobacterium tuberculosis isolates selected in pretomanid-treated mice, we discovered a novel resistance determinant, Rv2983, required for cofactor F420 biosynthesis and characterized the remarkable diversity of mutations in this and 5 other genes involved in nitroimidazole activation. We show that F420H2-deficient nitroimidazole-resistant mutants are hypersusceptible to the selective decontaminant malachite green used in solid media to isolate mycobacteria and may evade detection on such media. These results have important implications for development and clinical use of genotypic and phenotypic methods for nitroimidazole susceptibility testing.

microbiology↗