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ABSTRACTMacroautophagy/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.

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.

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.

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.

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.

Zika virus (ZIKV) is a significant global health threat, as infection has been linked to serious neurological complications, including microcephaly. Using a human stem cell-derived neural progenitor model system, we find that a critical cellular quality control process called the nonsense-mediated mRNA decay (NMD) pathway is disrupted during ZIKV infection. Importantly, disruption of the NMD pathway is a known cause of microcephaly and other neurological disorders. We further identify an interaction between the capsid protein of ZIKV and up-frameshift protein 1 (UPF1), the master regulator of NMD, and show that ZIKV capsid targets UPF1 for degradation. Together, these results offer a new mechanism for how ZIKV infection can cause neuropathology in the developing brain.

Therapeutic RNAs are stably expressed in blood cells of HIV-positive patients.