Biology subjects

Ryan, A. P.

Publications and source records attributed to Ryan, A. P..

8 recordsLinked to original sources

bioRxiv2023

Mitochondrial electron transport chain, ceramide and Coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, deficiency of coenzyme Q (CoQ), mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells results in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (under chow and high fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial Ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

cell biology↗

bioRxiv2022

Host specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including SARS-CoV-2, cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8s ability to sense coronavirus 3CLpros, and instead enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intra-species variation in inflammasome-mediated viral sensing and immunopathology.

immunology↗

bioRxiv2022

Antiviral function and viral antagonism of the rapidly evolving dynein activating adapter NINL

Viruses interact with the intracellular transport machinery to promote viral replication. Such host-virus interactions can drive host gene adaptation, leaving signatures of pathogen-driven evolution in host genomes. Here we leverage these genetic signatures to identify the dynein activating adaptor, ninein-like (NINL), as a critical component in the antiviral innate immune response and as a target of viral antagonism. Unique among genes encoding components of active dynein complexes, NINL has evolved under recurrent positive (diversifying) selection, particularly in its carboxy-terminal cargo binding region. Consistent with a role for NINL in host immunity, we demonstrate that NINL knockout cells exhibit an impaired response to interferon, resulting in increased permissiveness to viral replication. Moreover, we show that proteases encoded by diverse picornaviruses and coronaviruses cleave and disrupt NINL function in a host- and virus-specific manner. Our work reveals the importance of NINL in the antiviral response and the utility of using signatures of host-virus conflicts to uncover new components of antiviral immunity and targets of viral antagonism.

microbiology↗

bioRxiv2021

Resistance to miltefosine results from amplification of the RTA3 floppase or inactivation of flippases in Candida parapsilosis

Flippases and floppases are two classes of proteins that have opposing functions in the maintenance of lipid asymmetry of the plasma membrane. Flippases translocate lipids from the exoplasmic leaflet to the cytosolic leaflet, and floppases act in the opposite direction. Phosphatidylcholine (PC) is a major component of the eukaryotic plasma membrane and is asymmetrically distributed, being more abundant in the exoplasmic leaflet. Here we show that gene amplification of a putative PC floppase or double disruption of two PC flippases in the pathogenic yeast Candida parapsilosis results in resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism that has recently been granted orphan drug designation approval by the US FDA for treatment of invasive candidiasis. We analysed the genomes of 170 C. parapsilosis isolates and found that 107 of them have copy number variations (CNVs) at the RTA3 gene. RTA3 encodes a putative PC floppase whose deletion is known to increase the inward translocation of PC in Candida albicans. RTA3 copy number ranges from 2 to >40 across the C. parapsilosis isolates. Interestingly, 16 distinct CNVs with unique endpoints were identified, and phylogenetic analysis shows that almost all of them have originated only once. We found that increased copy number of RTA3 correlates with miltefosine resistance. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine over 26 days. Two genes, CPAR2_303950 and CPAR2_102700, gained homozygous protein-disrupting mutations in the evolved strains and code for putative PC flippases homologous to S. cerevisiae DNF1. Our results indicate that alteration of lipid asymmetry across the plasma membrane is a key mechanism of miltefosine resistance. We also find that C. parapsilosis is likely to gain resistance to miltefosine rapidly, because many isolates carry loss-of-function alleles in one of the flippase genes. Author summaryMiltefosine was developed as an anticancer drug but is commonly used to treat infections with the protozoan parasites Leishmania and Trypanosoma cruzi. More recently, it has been used to treat fungal infections, and in 2021 it was designated as an orphan drug by the US Food and Drug Administration for treatment of invasive candidiasis. Miltefosine is a derivative of phosphatidylcholine (PC), a major constituent of the cell membrane. PC and other phospholipids are asymmetrically distributed across the cell membrane. The mechanism of action of miltefosine is unknown. Here, we show that either increasing the activity of a putative floppase, which controls outward "flop" movement of phospholipids, or decreasing the activity of flippases, which control inward "flip" movement, results in increased resistance of the fungal pathogen Candida parapsilosis to miltefosine. This result suggests that miltefosine acts by controlling the localisation of PC or other phospholipids in the membrane. Importantly, we find that many C. parapsilosis isolates carry mutations in one flippase gene, which renders them partially resistant to miltefosine, and prone to easily acquiring increased resistance.

genomics↗

bioRxiv2020

Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection

Ribosomes are highly abundant cellular machines that perform the essential task of translating the genetic code into proteins. Cellular translation activity is finely tuned and proteostasis insults, such as those incurred upon viral infection, activate stress signaling pathways that result in translation reprogramming. Viral infection selectively shuts down host mRNA while redistributing ribosomes for selective translation of viral mRNAs. The intricacies of this selective ribosome shuffle from host to viral mRNAs are poorly understood. Here, we uncover a role for the ribosome associated quality control (RQC) factor ZNF598, a sensor for collided ribosomes, as a critical factor for vaccinia virus mRNA translation. Collided ribosomes are sensed by ZNF598, which ubiquitylates 40S subunit proteins uS10 and eS10 and thereby initiates RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection in human cells enhances uS10 ubiquitylation indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells which either lack ZNF598 or contain a ubiquitylation deficient version of uS10. Using SILAC-based proteomics and concurrent RNAseq analysis, we determine that host translation of vaccinia virus mRNAs is compromised in cells that lack RQC activity as compared to control cells whereas there was little evidence of differences in host or viral transcription. Additionally, vaccinia virus infection resulted in a loss of cellular RQC activity, suggesting that ribosomes engaged in viral protein production recruit ZNF598 away from its function in host translation. Thus, co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.

molecular biology↗

bioRxiv2020

Diverse viral proteases activate the NLRP1 inflammasome

The NLRP1 inflammasome is a multiprotein complex that is a potent activator of inflammation. Mouse NLRP1B can be activated through proteolytic cleavage by the bacterial Lethal Toxin (LeTx) protease, resulting in degradation of the N-terminal domains of NLRP1B and liberation of the bioactive C-terminal domain, which includes the caspase activation and recruitment domain (CARD). However, a natural pathogen-derived effector that can activate human NLRP1 remains unknown. Here, we use an evolutionary model to identify several proteases from diverse picornaviruses that cleave human NLRP1 within a rapidly evolving region of the protein, leading to host-specific and virus-specific activation of the NLRP1 inflammasome. Our work demonstrates that NLRP1 acts as a "tripwire" to recognize the enzymatic function of a wide range of viral proteases, and suggests that host mimicry of viral polyprotein cleavage sites can be an evolutionary strategy to activate a robust inflammatory immune response.

microbiology↗

bioRxiv2020

Cell cycle-gated feedback control mediates desensitization to interferon stimulation

Cells use sophisticated molecular circuits to interpret and respond to extracellular signal factors, such as hormones and cytokines, which are often released in a temporally varying fashion. In this study, we focus on type I interferon (IFN) signaling in human epithelial cells and combine microfluidics, time-lapse microscopy, and computational modeling to investigate how the IFN-responsive regulatory network operates in single cells to process repetitive IFN stimulation. We found that IFN- pretreatments lead to opposite effects, priming versus desensitization, depending on the input durations. These effects are governed by a regulatory network composed of a fast-acting positive feedback loop and a delayed negative feedback loop, mediated by upregulation of ubiquitin-specific peptidase 18 (USP18). We further revealed that USP18 upregulation can only be initiated at the G1 and early S phases of cell cycle upon the treatment onset, resulting in heterogeneous and delayed induction kinetics in single cells. This cell cycle gating provides a temporal compartmentalization of feedback control processes, enabling duration-dependent desensitization to repetitive stimulations. Moreover, our results, highlighting the importance of IFN dynamics, may suggest time-based strategies for enhancing the effectiveness of IFN pretreatment in clinical applications against viruses, such as SARS-CoV-2.

systems biology↗

bioRxiv2019

Tetrad analysis without tetrad dissection: Meiotic recombination and genomic diversity in the yeast Komagataella phaffii (Pichia pastoris)

Komagataella phaffii is a yeast widely used in the pharmaceutical and biotechnology industries, and is one of the two species that were formerly called Pichia pastoris. However, almost all laboratory work on K. phaffii has been done on strains derived from a single natural isolate, CBS7435. There is little information about the genetic properties of K. phaffii or its sequence diversity. Genetic analysis is difficult because, although K. phaffii makes asci with four spores, the spores are small and tend to clump together, making the asci hard to dissect. Here, we sequenced the genomes of all the known isolates of this species, and find that K. phaffii has only been isolated from nature four times. We analyzed the meiotic recombination landscape in a cross between auxotrophically marked strains derived from two isolates that differ at 44,000 single nucleotide polymorphism sites. We conducted tetrad analysis by making use of the property that haploids of this species do not mate in rich media, which enabled us to isolate and sequence the four types of haploid cell that are present in the colony that forms when a tetratype ascus germinates. We found that approximately 25 crossovers occur per meiosis, which is 3.5 times fewer than in Saccharomyces cerevisiae. Recombination is suppressed, and genetic diversity among natural isolates is low, in a region around centromeres that is much larger than the centromeres themselves. Our method of tetrad analysis without tetrad dissection will be applicable to other species whose spores do not mate spontaneously after germination.\n\nAuthor summaryTo better understand the basic genetics of the budding yeast Komagataella phaffii, which has many applications in biotechnology, we investigated its genetic diversity and its meiotic recombination landscape. We made a genetic cross between strains derived from two natural isolates, and developed a method for characterizing the genomes of the four spores resulting from meiosis, which were previously impossible to isolate. We found that K. phaffii has a lower recombination rate than Saccharomyces cerevisiae. It shows a large zone of suppressed recombination around its centromeres, which may be due to the structural differences between centromeres in K. phaffii and S. cerevisiae.

genetics↗