A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations.

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as an approximation for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays identified substitutions which conferred strong nirmatrelvir resistance and others that compromised activity. On the other hand, N142A and the P132H mutation, carried by the Omicron variant, caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

Residues forming the gating regions of asymmetric multidrug transporter Pdr5 also play roles in conformational switching and protein folding.

ATP-binding cassette (ABC) multidrug transporters are large, polytopic membrane proteins that exhibit astonishing promiscuity for their transport substrates. These transporters unidirectionally efflux thousands of structurally and functionally distinct compounds. To preclude the reentry of xenobiotic molecules via the drug-binding pocket, these proteins contain a highly conserved molecular gate, essentially allowing the transporters to function as molecular diodes. However, the structure-function relationship of these conserved gates and gating regions are not well characterized. In this study, we combine recent single-molecule, cryo-EM data with genetic and biochemical analyses of residues in the gating region of the yeast multidrug transporter Pdr5, the founding member of a large group of clinically relevant asymmetric ABC efflux pumps. Unlike the symmetric ABCG2 efflux gate, the Pdr5 counterpart is highly asymmetric, with only four (instead of six) residues comprising the gate proper. However, other residues in the near vicinity are essential for the gating activity. Furthermore, we demonstrate that residues in the gate and in the gating regions have multiple functions. For example, we show that Ile-685 and Val-1372 are required not only for successful efflux but also for allosteric inhibition of Pdr5 ATPase activity. Our investigations reveal that the gating region residues of Pdr5, and possibly other ABCG transporters, play a role not only in molecular gating but also in allosteric regulation, conformational switching, and protein folding.

mRNA detection in budding yeast with single fluorophores.

Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect >50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.

Robust, pleiotropic drug resistance 5 (Pdr5)-mediated multidrug resistance is vigorously maintained in Saccharomyces cerevisiae cells during glucose and nitrogen limitation.

Saccharomyces cerevisiae has sophisticated nutrient-sensing programs for responding to harsh environments containing limited nutrients. As a result, yeast cells can live in diverse environments, including animals, as a commensal or a pathogen. Because they live in mixed populations with other organisms that excrete toxic chemicals, it is of interest to know whether yeast cells maintain functional multidrug resistance mechanisms during nutrient stress. We measured the activity of Pdr5, the major Saccharomyces drug efflux pump under conditions of limiting nutrients. We demonstrate that the steady-state level of this transporter remains unchanged during growth in low concentrations of glucose and nitrogen even though two-dimensional gel electrophoresis revealed a decrease in the level of many proteins. We also evaluated rhodame 6G transport and resistance to three xenobiotic agents in rich (synthetic dextrose) and starvation medium. We demonstrate that Pdr5 function is vigorously maintained under both sets of conditions.

Structural dynamics of nucleosomes at single-molecule resolution.

The detailed mechanisms of how DNA that is assembled around a histone core can be accessed by DNA-binding proteins for transcription, replication, or repair, remain elusive nearly 40 years after Kornberg's nucleosome model was proposed. Uncovering the structural dynamics of nucleosomes is a crucial step in elucidating the mechanisms regulating genome accessibility. This requires the deconvolution of multiple structural states within an ensemble. Recent advances in single-molecule methods enable unprecedented efficiency in examining subpopulation dynamics. In this review, we summarize studies of nucleosome structure and dynamics from single-molecule approaches and how they advance our understanding of the mechanisms that govern DNA transactions.

Misregulation of Scm3p/HJURP causes chromosome instability in Saccharomyces cerevisiae and human cells.

The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability.

Clinical Correlation of Transcription Factor SOX3 in Cancer: Unveiling Its Role in Tumorigenesis.

Members of the SOX (SRY-related HMG box) family of transcription factors are crucial for embryonic development and cell fate determination. This review investigates the role of SOX3 in cancer, as aberrations in SOX3 expression have been implicated in several cancers, including osteosarcoma, breast, esophageal, endometrial, ovarian, gastric, hepatocellular carcinomas, glioblastoma, and leukemia. These dysregulations modulate key cancer outcomes such as apoptosis, epithelial-mesenchymal transition (EMT), invasion, migration, cell cycle, and proliferation, contributing to cancer development. SOX3 exhibits varied expression patterns correlated with clinicopathological parameters in diverse tumor types. This review aims to elucidate the nuanced role of SOX3 in tumorigenesis, correlating its expression with clinical and pathological characteristics in cancer patients and cellular modelsBy providing a comprehensive exploration of SOX3 involvement in cancer, this review underscores the multifaceted role of SOX3 across distinct tumor types. The complexity uncovered in SOX3 function emphasizes the need for further research to unravel its full potential in cancer therapeutics.

Insights into assembly and regulation of centromeric chromatin in Saccharomyces cerevisiae.

At the core of chromosome segregation is the centromere, which nucleates the assembly of a macromolecular kinetochore (centromere DNA and associated proteins) complex responsible for mediating spindle attachment. Recent advances in centromere research have led to identification of many kinetochore components, such as the centromeric-specific histone H3 variant, CenH3, and its interacting partner, Scm3. Both are essential for chromosome segregation and are evolutionarily conserved from yeast to humans. CenH3 is proposed to be the epigenetic mark that specifies centromeric identity. Molecular mechanisms that regulate the assembly of kinetochores at specific chromosomal sites to mediate chromosome segregation are not fully understood. In this review, we summarize the current literature and discuss results from our laboratory, which show that restricting the localization of budding yeast CenH3, Cse4, to centromeres and balanced stoichiometry between Scm3 and Cse4, contribute to faithful chromosome transmission. We highlight our findings that, similar to other eukaryotic centromeres, budding yeast centromeric histone H4 is hypoacetylated, and we discuss how altered histone acetylation affects chromosome segregation. This article is part of a Special Issue entitled: Chromatin in time and space.

A proteomic and transcriptomic approach reveals new insight into beta-methylthiolation of Escherichia coli ribosomal protein S12.

ß-methylthiolation is a novel post-translational modification mapping to a universally conserved Asp 88 of the bacterial ribosomal protein S12. This S12 specific modification has been identified on orthologs from multiple bacterial species. The origin and functional significance was investigated with both a proteomic strategy to identify candidate S12 interactors and expression microarrays to search for phenotypes that result from targeted gene knockouts of select candidates. Utilizing an endogenous recombinant E. coli S12 protein with an affinity tag as bait, mass spectrometric analysis identified candidate S12 binding partners including RimO (previously shown to be required for this post-translational modification) and YcaO, a conserved protein of unknown function. Transcriptomic analysis of bacterial strains with deleted genes for RimO and YcaO identified an overlapping transcriptional phenotype suggesting that YcaO and RimO likely share a common function. As a follow up, quantitative mass spectrometry additionally indicated that both proteins dramatically impacted the modification status of S12. Collectively, these results indicate that the YcaO protein is involved in ß-methylthiolation of S12 and its absence impairs the ability of RimO to modify S12. Additionally, the proteomic data from this study provides direct evidence that the E. coli specific ß-methylthiolation likely occurs when S12 is assembled as part of a ribosomal subunit.

Dissolvable alginate hydrogel-based biofilm microreactors for antibiotic susceptibility assays.

Biofilms are found in many infections in the forms of surface-adhering aggregates on medical devices, small clumps in tissues, or even in synovial fluid. Although antibiotic resistance genes are studied and monitored in the clinic, the structural and phenotypic changes that take place in biofilms can also lead to significant changes in how bacteria respond to antibiotics. Therefore, it is important to better understand the relationship between biofilm phenotypes and resistance and develop approaches that are compatible with clinical testing. Current methods for studying antimicrobial susceptibility are mostly planktonic or planar biofilm reactors. In this work, we develop a new type of biofilm reactor-three-dimensional (3D) microreactors-to recreate biofilms in a microenvironment that better mimics those in vivo where bacteria tend to form surface-independent biofilms in living tissues. The microreactors are formed on microplates, treated with antibiotics of 1000 times of the corresponding minimal inhibitory concentrations (1000 × MIC), and monitored spectroscopically with a microplate reader in a high-throughput manner. The hydrogels are dissolvable on demand without the need for manual scraping, thus enabling measurements of phenotypic changes. Bacteria inside the biofilm microreactors are found to survive exposure to 1000 × MIC of antibiotics, and subsequent comparison with plating results reveals no antibiotic resistance-associated phenotypes. The presented microreactor offers an attractive platform to study the tolerance and antibiotic resistance of surface-independent biofilms such as those found in tissues.

Naturally Occurring Mutations of SARS-CoV-2 Main Protease Confer Drug Resistance to Nirmatrelvir.

The SARS-CoV-2 main protease (Mpro) is the drug target of Pfizer's oral drug nirmatrelvir. The emergence of SARS-CoV-2 variants with mutations in Mpro raised the alarm of potential drug resistance. To identify potential clinically relevant drug-resistant mutants, we systematically characterized 102 naturally occurring Mpro mutants located at 12 residues at the nirmatrelvir-binding site, among which 22 mutations in 5 residues, including S144M/F/A/G/Y, M165T, E166 V/G/A, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (kcat/Km < 10-fold change) while being resistant to nirmatrelvir (Ki > 10-fold increase). X-ray crystal structures were determined for six representative mutants with and/or without GC-376/nirmatrelvir. Using recombinant SARS-CoV-2 viruses generated from reverse genetics, we confirmed the drug resistance in the antiviral assay and showed that Mpro mutants with reduced enzymatic activity had attenuated viral replication. Overall, our study identified several drug-resistant hotspots in Mpro that warrant close monitoring for possible clinical evidence of nirmatrelvir resistance, some of which have already emerged in independent viral passage assays conducted by others.

SARS-CoV-2 spike protein induces IL-18-mediated cardiopulmonary inflammation via reduced mitophagy.

Cardiopulmonary complications are major drivers of mortality caused by the SARS-CoV-2 virus. Interleukin-18, an inflammasome-induced cytokine, has emerged as a novel mediator of cardiopulmonary pathologies but its regulation via SARS-CoV-2 signaling remains unknown. Based on a screening panel, IL-18 was identified amongst 19 cytokines to stratify mortality and hospitalization burden in patients hospitalized with COVID-19. Supporting clinical data, administration of SARS-CoV-2 Spike 1 (S1) glycoprotein or receptor-binding domain (RBD) proteins into human angiotensin-converting enzyme 2 (hACE2) transgenic mice induced cardiac fibrosis and dysfunction associated with higher NF-κB phosphorylation (pNF-κB) and cardiopulmonary-derived IL-18 and NLRP3 expression. IL-18 inhibition via IL-18BP resulted in decreased cardiac pNF-κB and improved cardiac fibrosis and dysfunction in S1- or RBD-exposed hACE2 mice. Through in vivo and in vitro work, both S1 and RBD proteins induced NLRP3 inflammasome and IL-18 expression by inhibiting mitophagy and increasing mitochondrial reactive oxygenation species. Enhancing mitophagy prevented Spike protein-mediated IL-18 expression. Moreover, IL-18 inhibition reduced Spike protein-mediated pNF-κB and EC permeability. Overall, the link between reduced mitophagy and inflammasome activation represents a novel mechanism during COVID-19 pathogenesis and suggests IL-18 and mitophagy as potential therapeutic targets.

TCA and SSRI Antidepressants Exert Selection Pressure for Efflux-Dependent Antibiotic Resistance Mechanisms in Escherichia coli.

Microbial diversity is reduced in the gut microbiota of animals and humans treated with selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs). The mechanisms driving the changes in microbial composition, while largely unknown, is critical to understand considering that the gut microbiota plays important roles in drug metabolism and brain function. Using Escherichia coli, we show that the SSRI fluoxetine and the TCA amitriptyline exert strong selection pressure for enhanced efflux activity of the AcrAB-TolC pump, a member of the resistance-nodulation-cell division (RND) superfamily of transporters. Sequencing spontaneous fluoxetine- and amitriptyline-resistant mutants revealed mutations in marR and lon, negative regulators of AcrAB-TolC expression. In line with the broad specificity of AcrAB-TolC pumps these mutants conferred resistance to several classes of antibiotics. We show that the converse also occurs, as spontaneous chloramphenicol-resistant mutants displayed cross-resistance to SSRIs and TCAs. Chemical-genomic screens identified deletions in marR and lon, confirming the results observed for the spontaneous resistant mutants. In addition, deletions in 35 genes with no known role in drug resistance were identified that conferred cross-resistance to antibiotics and several displayed enhanced efflux activities. These results indicate that combinations of specific antidepressants and antibiotics may have important effects when both are used simultaneously or successively as they can impose selection for common mechanisms of resistance. Our work suggests that selection for enhanced efflux activities is an important factor to consider in understanding the microbial diversity changes associated with antidepressant treatments. IMPORTANCE Antidepressants are prescribed broadly for psychiatric conditions to alter neuronal levels of synaptic neurotransmitters such as serotonin and norepinephrine. Two categories of antidepressants are selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs); both are among the most prescribed drugs in the United States. While it is well-established that antidepressants inhibit reuptake of neurotransmitters there is evidence that they also impact microbial diversity in the gastrointestinal tract. However, the mechanisms and therefore biological and clinical effects remain obscure. We demonstrate antidepressants may influence microbial diversity through strong selection for mutant bacteria with increased AcrAB-TolC activity, an efflux pump that removes antibiotics from cells. Furthermore, we identify a new group of genes that contribute to cross-resistance between antidepressants and antibiotics, several act by regulating efflux activity, underscoring overlapping mechanisms. Overall, this work provides new insights into bacterial responses to antidepressants important for understanding antidepressant treatment effects.

Probing mutual interactions between Pseudomonas aeruginosa and Candida albicans in a biofabricated membrane-based microfluidic platform.

Microbes are typically found in multi-species (polymicrobial) communities. Cooperative and competitive interactions between species, mediated by diffusible factors and physical contact, leads to highly dynamic communities that undergo changes in composition diversity and size. Infections can be more severe or more difficult to treat when caused by multiple species. Interactions between species can improve the ability of one or more species to tolerate anti-microbial treatments and host defenses. Pseudomonas aeruginosa (Pa), a ubiquitous bacterium, and the opportunistic pathogenic yeast, Candida albicans (Ca), are frequently found together in cystic fibrosis lung infections and wound infections. While significant progress has been made in determining interactions between Pa and Ca, there are still important questions that remain unanswered. Here, we probe the mutual interactions between Pa and Ca in a custom-made microfluidic device using biopolymer chitosan membranes that support cross-species communication. By assembling microbes in physically separated, chemically communicating populations or bringing into direct interactions in a mixed culture, in situ polymicrobial growth and biofilm morphology were qualitatively characterized and quantified. Our work reveals new dynamic details of their mutual interactions including cooperation, competition, invasion, and biofilm formation. The membrane-based microfluidic platform can be further developed to understand the polymicrobial interactions within a controlled interactive microenvironment to improve microbial infection prevention and treatment.

A yeast-based system to study SARS-CoV-2 M pro structure and to identify nirmatrelvir resistant mutations.

The SARS-CoV-2 main protease (M pro ) is a major therapeutic target. The M pro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As M pro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for M pro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of M pro E166R, and M pro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of M pro that may arise as M pro antivirals become more widely used.

Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir.

The SARS-CoV-2 main protease (M pro ) is the drug target of Pfizer’s oral drug Paxlovid. The emergence of SARS-CoV-2 variants with mutations in M pro raised the alarm of potential drug resistance. In this study, we identified 100 naturally occurring M pro mutations located at the nirmatrelvir binding site, among which 20 mutants, including S144M/F/A/G/Y, M165T, E166G, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (k cat /K m <10-fold change) and resistance to nirmatrelvir (K i >10-fold increase). X-ray crystal structures were determined for seven representative mutants with and/or without GC-376/nirmatrelvir. Viral growth assay showed that M pro mutants with reduced enzymatic activity led to attenuated viral replication. Overall, our study identified several drug resistant hot spots that warrant close monitoring for possible clinical evidence of Paxlovid resistance. One Sentence Summary: Paxlovid resistant SARS-CoV-2 viruses with mutations in the main protease have been identified from clinical isolates.

A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations.

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

Eltrombopag Improves Erythroid Differentiation in a Human Induced Pluripotent Stem Cell Model of Diamond Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is a congenital macrocytic anemia associated with ribosomal protein haploinsufficiency. Ribosomal dysfunction delays globin synthesis, resulting in excess toxic free heme in erythroid progenitors, early differentiation arrest, and pure red cell aplasia. In this study, DBA induced pluripotent stem cell (iPSC) lines were generated from blood mononuclear cells of DBA patients with inactivating mutations in RPS19 and subjected to hematopoietic differentiation to model disease phenotypes. In vitro differentiated hematopoietic cells were used to investigate whether eltrombopag, an FDA-approved mimetic of thrombopoietin with robust intracellular iron chelating properties, could rescue erythropoiesis in DBA by restricting the labile iron pool (LIP) derived from excessive free heme. DBA iPSCs exhibited RPS19 haploinsufficiency, reduction in the 40S/60S ribosomal subunit ratio and early erythroid differentiation arrest in the absence of eltrombopag, compared to control isogenic iPSCs established by CRISPR/Cas9-mediated correction of the RPS19 point mutation. Notably, differentiation of DBA iPSCs in the presence of eltrombopag markedly improved erythroid maturation. Consistent with a molecular mechanism based on intracellular iron chelation, we observed that deferasirox, a clinically licensed iron chelator able to permeate into cells, also enhanced erythropoiesis in our DBA iPSC model. In contrast, erythroid maturation did not improve substantially in DBA iPSC differentiation cultures supplemented with deferoxamine, a clinically available iron chelator that poorly accesses LIP within cellular compartments. These findings identify eltrombopag as a promising new therapeutic to improve anemia in DBA.

DNA methylation increases nucleosome compaction and rigidity.

Cytosine methylation on CpG dinucleotides is an essential epigenetic modification in eukaryotes. How DNA methylation modulates nucleosome structure and dynamics has been a long-standing question. We implemented a single-molecule method to monitor the effects of DNA methylation on the structure and dynamics of mononucleosomes. Our studies show that DNA methylation induces a more compact and rigid nucleosome structure, providing a physical basis for how DNA methylation might contribute to regulating chromatin structure.