Sandoval, C. | bioRxiv Science

bioRxiv2020

Precise Estimation of In Vivo Protein Turnover Rates

Isotopic labeling with deuterium oxide (D2O) is a common technique for estimating in vivo protein turnover, but its use has been limited by two long-standing problems: (1) identifying non-monoisotopic peptides; and (2) estimating protein turnover rates in the presence of dynamic amino acid enrichment. In this paper, we present a novel experimental and analytical framework for solving these two problems. Peptides with high probabilities of labeling in many amino acids present fragmentation spectra that frequently do not match the theoretical spectra used in standard identification algorithms. We resolve this difficulty using a modified search algorithm we call Conditional Ion Distribution Search (CIDS). Increased identifications from CIDS along with direct measurement of amino acid enrichment and statistical modeling that accounts for heterogeneous information across peptides, dramatically improves the accuracy and precision of half-life estimates. We benchmark the approach in cells, where near-complete labeling is possible, and conduct an in vivo experiment revealing, for the first time, differences in protein turnover between mice and naked mole-rats commensurate with their disparate longevity.

bioinformatics↗

bioRxiv2020

HIV Vpr modulates the host DNA damage response at two independent steps to damage DNA and repress double-strand DNA break repair

The DNA damage response (DDR) is a signaling cascade that is vital to ensuring the fidelity of the host genome in the presence of genotoxic stress. Growing evidence has emphasized the importance of both activation and repression of the host DDR by diverse DNA and RNA viruses. Previous work has shown that HIV-1 is also capable of engaging the host DDR, primarily through the conserved accessory protein Vpr. However, the extent of this engagement has remained unclear. Here we show that HIV-1 and HIV-2 Vpr directly induce DNA damage and stall DNA replication, leading to the activation of several markers of double- and single-strand DNA breaks. Despite causing damage and activating the DDR, we found that Vpr repress the repair of double-strand breaks (DSB) by inhibiting homologous recombination (HR) and non-homologous end joining (NHEJ). Mutational analyses of Vpr revealed that DNA damage and DDR activation are independent from repression of HR and Vpr-mediated cell-cycle arrest. Moreover, we show that repression of HR does not require cell-cycle arrest but instead may precede this long-standing enigmatic Vpr phenotype. Together, our data uncover that Vpr globally modulates the host DDR at at least two independent steps; offering novel insight into the primary functions of lentiviral Vpr and the roles of the DNA damage response in lentiviral replication. IMPORTANCEThe DNA damage response (DDR) is a signaling cascade that safeguards the genome from genotoxic agents, including human pathogens. However, the DDR has also been utilized by many pathogens, such as Human Immunodeficiency Virus (HIV), to enhance infection. To properly treat HIV positive individuals, we must understand how the virus usurps our own cellular processes. Here, we have found that an important yet poorly-understood gene in HIV, Vpr, targets the DDR at two unique steps: it causes damage and activates DDR signaling, and it represses the ability of cells to repair this damage, which we hypothesize is central to the primary function of Vpr. In clarifying these important functions of Vpr, our work highlights the multiple ways human pathogens engage the DDR, and further suggests that modulation of the DDR may be a novel way to help in the fight against HIV.

microbiology↗