Molecular functions of ANKLE2 and its implications in human disease.

Ankyrin repeat and LEM domain-containing 2 (ANKLE2) is a scaffolding protein with established roles in cell division and development, the dysfunction of which is increasingly implicated in human disease. ANKLE2 regulates nuclear envelope disassembly at the onset of mitosis and its reassembly after chromosome segregation. ANKLE2 dysfunction is associated with abnormal nuclear morphology and cell division. It regulates the nuclear envelope by mediating protein-protein interactions with barrier to autointegration factor (BANF1; also known as BAF) and with the kinase and phosphatase that modulate the phosphorylation state of BAF. In brain development, ANKLE2 is crucial for proper asymmetric division of neural progenitor cells. In humans, pathogenic loss-of-function mutations in ANKLE2 are associated with primary congenital microcephaly, a condition in which the brain is not properly developed at birth. ANKLE2 is also linked to other disease pathologies, including congenital Zika syndrome, cancer and tauopathy. Here, we review the molecular roles of ANKLE2 and the recent literature on human diseases caused by its dysfunction.

I'm Walking into Spiderwebs: Making Sense of Protein-Protein Interaction Data.

Protein-protein interactions (PPIs) are at the heart of the molecular landscape permeating life. Proteomics studies can explore this protein interaction landscape using mass spectrometry (MS). Thanks to their high sensitivity, mass spectrometers can easily identify thousands of proteins within a single sample, but that same sensitivity generates tangled spiderwebs of data that hide biologically relevant findings. So, what does a researcher do when she finds herself walking into spiderwebs? In a field focused on discovery, MS data require rigor in their analysis, experimental validation, or a combination of both. In this Review, we provide a brief primer on MS-based experimental methods to identify PPIs. We discuss approaches to analyze the resulting data and remove the proteomic background. We consider the advantages between comprehensive and targeted studies. We also discuss how scoring might be improved through AI-based protein structure information. Women have been essential to the development of proteomics, so we will specifically highlight work by women that has made this field thrive in recent years.

A Zika virus protein expression screen in Drosophila to investigate targeted host pathways during development.

In the past decade, Zika virus (ZIKV) emerged as a global public health concern. Although adult infections are typically mild, maternal infection can lead to adverse fetal outcomes. Understanding how ZIKV proteins disrupt development can provide insights into the molecular mechanisms of disease caused by this virus, which includes microcephaly. In this study, we generated a toolkit to ectopically express ZIKV proteins in vivo in Drosophila melanogaster in a tissue-specific manner using the GAL4/UAS system. We used this toolkit to identify phenotypes and potential host pathways targeted by the virus. Our work identified that expression of most ZIKV proteins caused scorable phenotypes, such as overall lethality, gross morphological defects, reduced brain size and neuronal function defects. We further used this system to identify strain-dependent phenotypes that may have contributed to the increased pathogenesis associated with the outbreak of ZIKV in the Americas in 2015. Our work demonstrates the use of Drosophila as an efficient in vivo model to rapidly decipher how pathogens cause disease and lays the groundwork for further molecular study of ZIKV pathogenesis in flies.

Computational evaluation of light propagation in cylindrical bioreactors for optogenetic mammalian cell cultures.

Light-inducible regulation of cellular pathways and gene circuits in mammalian cells is a new frontier in mammalian genetic engineering. Optogenetic mammalian cell cultures, which are light-sensitive engineered cells, utilize light to regulate gene expression and protein activity. As a low-cost, tunable, and reversible input, light is highly adept at spatiotemporal and orthogonal regulation of cellular behavior. However, light is absorbed and scattered as it travels through media and cells, and the applicability of optogenetics in larger mammalian bioreactors has not been determined. In this work, we computationally explore the size limit to which optogenetics can be applied in cylindrical bioreactors at relevant height-to-diameter ratios. We model the propagation of light using the radiative transfer equation and consider changes in reactor volume, absorption coefficient, scattering coefficient, and scattering anisotropy. We observe sufficient light penetration for activation in simulated bioreactors with sizes of up to 80,000 L at maximal cell densities. We performed supporting experiments and found that significant attenuation occurs at the boundaries of the system, but the relative change in intensity distribution within the reactor was consistent with simulation results. We conclude that optogenetics can be applied to bioreactors at an industrial scale and may be a valuable tool for specific biomanufacturing applications.

A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2 deficient pancreatic cancer.

Pancreatic cancer is one of the deadliest diseases in human malignancies. Among total pancreatic cancer patients, ~10% of patients are categorized as familial pancreatic cancer (FPC) patients, carrying germline mutations of the genes involved in DNA repair pathways (e.g., BRCA2). Personalized medicine approaches tailored toward patients' mutations would improve patients' outcome. To identify novel vulnerabilities of BRCA2-deficient pancreatic cancer, we generated isogenic Brca2-deficient murine pancreatic cancer cell lines and performed high-throughput drug screens. High-throughput drug screening revealed that Brca2-deficient cells are sensitive to Bromodomain and Extraterminal Motif (BET) inhibitors, suggesting that BET inhibition might be a potential therapeutic approach. We found that BRCA2 deficiency increased autophagic flux, which was further enhanced by BET inhibition in Brca2-deficient pancreatic cancer cells, resulting in autophagy-dependent cell death. Our data suggests that BET inhibition can be a novel therapeutic strategy for BRCA2-deficient pancreatic cancer.

A Zika virus protein expression screen in Drosophila to investigate targeted host pathways during development.

In the past decade, Zika virus (ZIKV) emerged as a global public health concern. While adult infections are typically mild, maternal infection can lead to adverse fetal outcomes. Understanding how ZIKV proteins disrupt development can provide insights into the molecular mechanisms of symptoms caused by this virus including microcephaly. In this study, we generated a toolkit to ectopically express Zika viral proteins in vivo in Drosophila melanogaster in a tissue-specific manner using the GAL4/UAS system. We use this toolkit to identify phenotypes and host pathways targeted by the virus. Our work identified that expression of most ZIKV proteins cause scorable phenotypes, such as overall lethality, gross morphological defects, reduced brain size, and neuronal function defects. We further use this system to identify strain-dependent phenotypes that may contribute to the increased pathogenesis associated with the more recent outbreak of ZIKV in the Americas. Our work demonstrates Drosophila's use as an efficient in vivo model to rapidly decipher how pathogens cause disease and lays the groundwork for further molecular study of ZIKV pathogenesis in flies.

Dengue virus NS5 degrades ERC1 during infection to antagonize NF-kB activation.

Dengue virus (DENV) is the most important human virus transmitted by mosquitos. Dengue pathogenesis is characterized by a large induction of proinflammatory cytokines. This cytokine induction varies among the four DENV serotypes (DENV1 to 4) and poses a challenge for live DENV vaccine design. Here, we identify a viral mechanism to limit NF-κB activation and cytokine secretion by the DENV protein NS5. Using proteomics, we found that NS5 binds and degrades the host protein ERC1 to antagonize NF-κB activation, limit proinflammatory cytokine secretion, and reduce cell migration. We found that ERC1 degradation involves unique properties of the methyltransferase domain of NS5 that are not conserved among the four DENV serotypes. By obtaining chimeric DENV2 and DENV4 viruses, we map the residues in NS5 for ERC1 degradation, and generate recombinant DENVs exchanging serotype properties by single amino acid substitutions. This work uncovers a function of the viral protein NS5 to limit cytokine production, critical to dengue pathogenesis. Importantly, the information provided about the serotype-specific mechanism for counteracting the antiviral response can be applied to improve live attenuated vaccines.

The Archer and the Prey: The Duality of PAF1C in Antiviral Immunity.

In the ongoing arms race between virus and host, fine-tuned gene expression plays a critical role in antiviral signaling. However, viruses have evolved to disrupt this process and promote their own replication by targeting host restriction factors. Polymerase-associated factor 1 complex (PAF1C) is a key player in this relationship, recruiting other host factors to regulate transcription and modulate innate immune gene expression. Consequently, PAF1C is consistently targeted by a diverse range of viruses, either to suppress its antiviral functions or co-opt them for their own benefit. In this review, we delve into the current mechanisms through which PAF1C restricts viruses by activating interferon and inflammatory responses at the transcriptional level. We also highlight how the ubiquity of these mechanisms makes PAF1C especially vulnerable to viral hijacking and antagonism. Indeed, as often as PAF1C is revealed to be a restriction factor, viruses are found to have targeted the complex in reply.

Polyamines and eIF5A hypusination facilitate SREBP2 synthesis and cholesterol production leading to enhanced enterovirus attachment and infection.

Metabolism is key to cellular processes that underlie the ability of a virus to productively infect. Polyamines are small metabolites vital for many host cell processes including proliferation, transcription, and translation. Polyamine depletion also inhibits virus infection via diverse mechanisms, including inhibiting polymerase activity and viral translation. We showed that Coxsackievirus B3 (CVB3) attachment requires polyamines; however, the mechanism was unknown. Here, we report polyamines' involvement in translation, through a process called hypusination, promotes expression of cholesterol synthesis genes by supporting SREBP2 synthesis, the master transcriptional regulator of cholesterol synthesis genes. Measuring bulk transcription, we find polyamines support expression of cholesterol synthesis genes, regulated by SREBP2. Thus, polyamine depletion inhibits CVB3 by depleting cellular cholesterol. Exogenous cholesterol rescues CVB3 attachment, and mutant CVB3 resistant to polyamine depletion exhibits resistance to cholesterol perturbation. This study provides a novel link between polyamine and cholesterol homeostasis, a mechanism through which polyamines impact CVB3 infection.

Nuclear accumulation of host transcripts during Zika Virus Infection.

Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.

Quantitative and temporal measurement of dynamic autophagy rates.

Macroautophagy/autophagy is a multistep degradative process that is essential for maintaining cellular homeostasis and is often dysregulated during disease. Systematically quantifying flux through this pathway is critical for gaining fundamental insights and effectively modulating this process. Established methods to quantify flux use steady-state measurements, which provide limited information about the perturbation and the cellular response. We present a theoretical and experimental framework to measure autophagic steps in the form of rates under non-steady-state conditions. We use this approach to measure temporal responses to rapamycin and wortmannin treatments, two commonly used autophagy modulators. We quantified changes in autophagy rates in as little as 10 min, which can establish direct mechanisms for autophagy perturbation before feedback begins. We identified concentration-dependent effects of rapamycin on the initial and temporal progression of autophagy rates. We also found variable recovery time from wortmannin's inhibition of autophagy, which is further accelerated by rapamycin. Furthermore, we applied this approach to study the effect of serum and glutamine starvation on autophagy. Serum starvation led to a rapid and transient increase in all the rates. Glutamine starvation led to a decrease in the rates on a longer timescale. In summary, this new approach enables the quantification of autophagy flux with high sensitivity and temporal resolution and facilitates a comprehensive understanding of this process.

Transcriptomic profiling implicates PAF1 in both active and repressive immune regulatory networks.

BACKGROUND: Sitting at the interface of gene expression and host-pathogen interaction, polymerase associated factor 1 complex (PAF1C) is a rising player in the innate immune response. The complex localizes to the nucleus and associates with chromatin to modulate RNA polymerase II (RNAPII) elongation of gene transcripts. Performing this function at both proximal and distal regulatory elements, PAF1C interacts with many host factors across such sites, along with several microbial proteins during infection. Therefore, translating the ubiquity of PAF1C into specific impacts on immune gene expression remains especially relevant. RESULTS: Advancing past work, we treat PAF1 knockout cells with a slate of immune stimuli to identify key trends in PAF1-dependent gene expression with broad analytical depth. From our transcriptomic data, we confirm PAF1 is an activator of traditional immune response pathways as well as other cellular pathways correlated with pathogen defense. With this model, we employ computational approaches to refine how PAF1 may contribute to both gene activation and suppression. Specifically focusing on transcriptional motifs and regulons, we predict gene regulatory elements strongly associated with PAF1, including those implicated in an immune response. Overall, our results suggest PAF1 is involved in innate immunity at several distinct axes of regulation. CONCLUSIONS: By identifying PAF1-dependent gene expression across several pathogenic contexts, we confirm PAF1C to be a key mediator of innate immunity. Combining these transcriptomic profiles with potential regulatory networks corroborates the previously identified functions of PAF1C. With this, we foster new avenues for its study as a regulator of innate immunity, and our results will serve as a basis for targeted study of PAF1C in future validation studies.

Hippo signaling pathway activation during SARS-CoV-2 infection contributes to host antiviral response.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, causes respiratory failure and damage to multiple organ systems. The emergence of viral variants poses a risk of vaccine failures and prolongation of the pandemic. However, our understanding of the molecular basis of SARS-CoV-2 infection and subsequent COVID-19 pathophysiology is limited. In this study, we have uncovered a critical role for the evolutionarily conserved Hippo signaling pathway in COVID-19 pathogenesis. Given the complexity of COVID-19-associated cell injury and immunopathogenesis processes, we investigated Hippo pathway dynamics in SARS-CoV-2 infection by utilizing COVID-19 lung samples and human cell models based on pluripotent stem cell-derived cardiomyocytes (PSC-CMs) and human primary lung air-liquid interface (ALI) cultures. SARS-CoV-2 infection caused activation of the Hippo signaling pathway in COVID-19 lung and in vitro cultures. Both parental and Delta variant of concern (VOC) strains induced Hippo pathway. The chemical inhibition and gene knockdown of upstream kinases MST1/2 and LATS1 resulted in significantly enhanced SARS-CoV-2 replication, indicating antiviral roles. Verteporfin, a pharmacological inhibitor of the Hippo pathway downstream transactivator, YAP, significantly reduced virus replication. These results delineate a direct antiviral role for Hippo signaling in SARS-CoV-2 infection and the potential for this pathway to be pharmacologically targeted to treat COVID-19.

Production of novel SARS-CoV-2 Spike truncations in Chinese hamster ovary cells leads to high expression and binding to antibodies.

SARS-CoV-2 Spike is a key protein that mediates viral entry into cells and elicits antibody responses. Its importance in infection, diagnostics, and vaccinations has created a large demand for purified Spike for clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, the Spike receptor-binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg L-1 on day 7. In comparison, RBD titers peak at 54 mg L-1 on day 3. Next, we develop eight Spike truncations (T1-T8) in pursuit of truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 and 73 mg L-1 , respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest that T1 is a promising Spike alternative for use in various applications.

Systems Biology of Virus-Host Protein Interactions: From Hypothesis Generation to Mechanisms of Replication and Pathogenesis.

As obligate intracellular parasites, all viruses must co-opt cellular machinery to facilitate their own replication. Viruses often co-opt these cellular pathways and processes through physical interactions between viral and host proteins. In addition to facilitating fundamental aspects of virus replication cycles, these virus-host protein interactions can also disrupt physiological functions of host proteins, causing disease that can be advantageous to the virus or simply a coincidence. Consequently, unraveling virus-host protein interactions can serve as a window into molecular mechanisms of virus replication and pathogenesis. Identifying virus-host protein interactions using unbiased systems biology approaches provides an avenue for hypothesis generation. This review highlights common systems biology approaches for identification of virus-host protein interactions and the mechanistic insights revealed by these methods. We also review conceptual innovations using comparative and integrative systems biology that can leverage global virus-host protein interaction data sets to more rapidly move from hypothesis generation to mechanism.

Hippo Signaling Pathway Activation during SARS-CoV-2 Infection Contributes to Host Antiviral Response.

SARS-CoV-2, responsible for the COVID-19 pandemic, causes respiratory failure and damage to multiple organ systems. The emergence of viral variants poses a risk of vaccine failures and prolongation of the pandemic. However, our understanding of the molecular basis of SARS-CoV-2 infection and subsequent COVID-19 pathophysiology is limited. In this study, we have uncovered a critical role for the evolutionarily conserved Hippo signaling pathway in COVID-19 pathogenesis. Given the complexity of COVID-19 associated cell injury and immunopathogenesis processes, we investigated Hippo pathway dynamics in SARS-CoV-2 infection by utilizing COVID-19 lung samples, and human cell models based on pluripotent stem cell-derived cardiomyocytes (PSC-CMs) and human primary lung air-liquid interface (ALI) cultures. SARS-CoV-2 infection caused activation of the Hippo signaling pathway in COVID-19 lung and in vitro cultures. Both parental and Delta variant of concern (VOC) strains induced Hippo pathway. The chemical inhibition and gene knockdown of upstream kinases MST1/2 and LATS1 resulted in significantly enhanced SARS-CoV-2 replication, indicating antiviral roles. Verteporfin a pharmacological inhibitor of the Hippo pathway downstream transactivator, YAP, significantly reduced virus replication. These results delineate a direct antiviral role for Hippo signaling in SARS-CoV-2 infection and the potential for this pathway to be pharmacologically targeted to treat COVID-19.

Let's Get Physical: Flavivirus-Host Protein-Protein Interactions in Replication and Pathogenesis.

Flaviviruses comprise a genus of viruses that pose a significant burden on human health worldwide. Transmission by both mosquito and tick vectors, and broad host tropism contribute to the presence of flaviviruses globally. Like all viruses, they require utilization of host molecular machinery to facilitate their replication through physical interactions. Their RNA genomes are translated using host ribosomes, synthesizing viral proteins that cooperate with each other and host proteins to reshape the host cell into a factory for virus replication. Thus, dissecting the physical interactions between viral proteins and their host protein targets is essential in our comprehension of how flaviviruses replicate and how they alter host cell behavior. Beyond replication, even single interactions can contribute to immune evasion and pathogenesis, providing potential avenues for therapeutic intervention. Here, we review protein interactions between flavivirus and host proteins that contribute to virus replication, immune evasion, and disease.

SARS-CoV-2 spike binding to ACE2 is stronger and longer ranged due to glycan interaction.

Highly detailed steered molecular dynamics simulations are performed on differently glycosylated receptor binding domains of the severe acute respiratory syndrome coronavirus-2 spike protein. The binding strength and the binding range increase with glycosylation. The interaction energy rises very quickly when pulling the proteins apart and only slowly drops at larger distances. We see a catch-slip-type behavior whereby interactions during pulling break and are taken over by new interactions forming. The dominant interaction mode is hydrogen bonds, but Lennard-Jones and electrostatic interactions are relevant as well.

Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes.

Dengue virus (DENV) disruption of the innate immune response is critical to establish infection. DENV non-structural protein 5 (NS5) plays a central role in this disruption, such as antagonism of STAT2. We recently found that DENV serotype 2 (DENV2) NS5 interacts with Polymerase associated factor 1 complex (PAF1C). The primary members of PAF1C are PAF1, LEO1, CTR9, and CDC73. This nuclear complex is an emerging player in the immune response. It promotes the expression of many genes, including genes related to the antiviral, antimicrobial and inflammatory responses, through close association with the chromatin of these genes. Our previous work demonstrated that NS5 antagonizes PAF1C recruitment to immune response genes. However, it remains unknown if NS5 antagonism of PAF1C is complementary to its antagonism of STAT2. Here, we show that knockout of PAF1 enhances DENV2 infectious virion production. By comparing gene expression profiles in PAF1 and STAT2 knockout cells, we find that PAF1 is necessary to express immune response genes that are STAT2-independent. Finally, we mapped the viral determinants for the NS5-PAF1C protein interaction. We found that NS5 nuclear localization and the C-terminal region of the methyltransferase domain are required for its interaction with PAF1C. Mutation of these regions rescued the expression of PAF1-dependent immune response genes that are antagonized by NS5. In sum, our results support a role for PAF1C in restricting DENV2 replication that NS5 antagonizes through its protein interaction with PAF1C.

Contributions of the international plant science community to the fight against infectious diseases in humans-part 2: Affordable drugs in edible plants for endemic and re-emerging diseases.

The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection.