INVESTIGATING THE ROLE OF POST-TRANSLATIONAL MODIFICATIONS IN THE CORE RAS GTPASE DOMAIN

Ras proteins are the most commonly mutated oncoproteins in cancer (~30%). Oncogenic, activating Ras mutations are known drivers of the deadliest human cancers, including lung, pancreatic and colorectal cancers. Ras proteins function as critical regulators of cellular growth by acting as molecular switches, cycling between active, GTP- and inactive, GDP-bound states. In their active form, Ras proteins signal through downstream pathways that regulate cellular growth, differentiation and apoptosis. Early attempts to target Ras proteins (farnesyltransferase inhibitors) were directed toward inhibiting key carboxyl (C)-terminal lipid post-translational modifications (PTMs), which are crucial for proper Ras localization and function at the cellular membrane. Despite their failure, FTIs represent the first direct targeting efforts of Ras proteins. Promising new classes of anti-cancer drugs directed at targeting the dysregulation of PTM status in cancers (kinase inhibitors, histone deacetylase inhibitors, HDACi and methyltransferase inhibitors) have demonstrated multiple clinical successes in recent years. PTMs have been demonstrated to alter protein stability and localization as well as protein-protein interactions in several non-histone cancer-related proteins. While PTMs have been extensively studied in the C-terminus of Ras proteins, their role remains poorly understood in the core Ras guanine nucleotide binding domain (GTPase domain). Monoubiquitylation and acetylation within the core Ras GTPase domain have been demonstrated to modulate Ras protein activity, signaling and tumorigenesis, suggesting that PTMs in this region are capable of regulating Ras behavior. Further, aberrant dysregulation in the balance of PTMs has been characterized in several cancer types, including the Ras-driven pancreatic cancer. It is therefore reasonable that Ras PTMs may present a novel avenue for therapeutic targeting in cancer. Despite more than three decades of research, Ras has remained an elusive target for cancer therapy. We have recently identified novel sites of PTMs in Ras proteins at highly conserved residues within the core GTPase domain. Herein, we present highly innovative and novel methods of generating both acetyl- and methyl-lysine in intact Ras proteins. With the combined use of biochemical, structural, cellular and computational data, we provide mechanistic insight into the regulation Ras proteins by PTMs and also provide rationale for novel therapeutic targeting approaches in Ras-driven cancers.Doctor of Philosoph

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity

Analysis of the Diet of the Shenandoah Valley Barn Owl

The barn owl or the Tyto alba is a nocturnal bird of prey. Barn owls are an important creature in the balancing and control of pest and rodent populations. Often being identified by their appearance, the common barn owl has a white or tan underside, long wings colored in shades of black, brown, tan and white, and a distinct heart-shaped white facial disk. The unique features of the barn owl\u27s face make them have excellent senses for hunting. The facial disk actually funnels the sound into the ear holes of the owl, which are located on either side of the face. The owl also can sense how far the prey object is away because of another unique feature, lopsided or uneven ear holes. Because of these features, the barn owl is well suited for its nocturnal hunting status. The main diet of the barn owl is mostly small mammals. Studies have shown that voles seem to be the most prevalent food source for the owl , but other small rodents such as mice, shrews, and moles are also consumed. Studies also show that over ninety-five percent of the diet of the barn owl consists of these small mammals . Other non-mammalian prey consisting of birds, amphibians, reptiles, and large insects is also eaten. However, the diet of the owls will depend greatly on the population of the rodents in the area. Because of the diet of the barn owl, they are useful in the natural management and control of many pest populations. The prey of the barn owl is often swallowed whole. If they prey is too large to consume in this way it is torn into small pieces. After ingestion, the indigestible portions of the prey such as the bones, teeth, and fur are regurgitated in what we refer to as an owl pellet . These pellets are often one to two inches in length and contain all of the indigestible portions of the owl\u27s diet. In this way, the pellet makes a perfect tool for analysis. Because it contains the bones of the owl\u27s prey, a diet analysis can be performed simply by analyzing the remains within the pellet. The primary diet of the owl can then be determined and statistical results can be drawn. These results can be compared to those from other geographical locations to see if there are deviations

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAScan be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cellbased properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains bothWTand mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity

Complete Genome Sequence of a Novel Coronavirus (SARS-CoV-2) Isolate from Bangladesh.

The complete genome sequence of a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) isolate obtained from a nasopharyngeal swab from a patient with COVID-19 in Bangladesh is reported

Complete Genome Sequence of a Novel Coronavirus (SARS-CoV-2) Isolate from Bangladesh.

The complete genome sequence of a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) isolate obtained from a nasopharyngeal swab from a patient with COVID-19 in Bangladesh is reported

Upper airway gene expression differentiates COVID-19 from other acute respiratory illnesses and reveals suppression of innate immune responses by SARS-CoV-2.

We studied the host transcriptional response to SARS-CoV-2 by performing metagenomic sequencing of upper airway samples in 238 patients with COVID-19, other viral or non-viral acute respiratory illnesses (ARIs). Compared to other viral ARIs, COVID-19 was characterized by a diminished innate immune response, with reduced expression of genes involved in toll-like receptor and interleukin signaling, chemokine binding, neutrophil degranulation and interactions with lymphoid cells. Patients with COVID-19 also exhibited significantly reduced proportions of neutrophils and macrophages, and increased proportions of goblet, dendritic and B-cells, compared to other viral ARIs. Using machine learning, we built 26-, 10- and 3-gene classifiers that differentiated COVID-19 from other acute respiratory illnesses with AUCs of 0.980, 0.950 and 0.871, respectively. Classifier performance was stable at low viral loads, suggesting utility in settings where direct detection of viral nucleic acid may be unsuccessful. Taken together, our results illuminate unique aspects of the host transcriptional response to SARS-CoV-2 in comparison to other respiratory viruses and demonstrate the feasibility of COVID-19 diagnostics based on patient gene expression

Upper airway gene expression reveals suppressed immune responses to SARS-CoV-2 compared with other respiratory viruses.

SARS-CoV-2 infection is characterized by peak viral load in the upper airway prior to or at the time of symptom onset, an unusual feature that has enabled widespread transmission of the virus and precipitated a global pandemic. How SARS-CoV-2 is able to achieve high titer in the absence of symptoms remains unclear. Here, we examine the upper airway host transcriptional response in patients with COVID-19 (n = 93), other viral (n = 41) or non-viral (n = 100) acute respiratory illnesses (ARIs). Compared with other viral ARIs, COVID-19 is characterized by a pronounced interferon response but attenuated activation of other innate immune pathways, including toll-like receptor, interleukin and chemokine signaling. The IL-1 and NLRP3 inflammasome pathways are markedly less responsive to SARS-CoV-2, commensurate with a signature of diminished neutrophil and macrophage recruitment. This pattern resembles previously described distinctions between symptomatic and asymptomatic viral infections and may partly explain the propensity for pre-symptomatic transmission in COVID-19. We further use machine learning to build 27-, 10- and 3-gene classifiers that differentiate COVID-19 from other ARIs with AUROCs of 0.981, 0.954 and 0.885, respectively. Classifier performance is stable across a wide range of viral load, suggesting utility in mitigating false positive or false negative results of direct SARS-CoV-2 tests

Community Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 Disproportionately Affects the Latinx Population During Shelter-in-Place in San Francisco.

BackgroundThere is an urgent need to understand the dynamics and risk factors driving ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission during shelter-in-place mandates.MethodsWe offered SARS-CoV-2 reverse-transcription polymerase chain reaction (PCR) and antibody (Abbott ARCHITECT IgG) testing, regardless of symptoms, to all residents (aged ≥4 years) and workers in a San Francisco census tract (population: 5174) at outdoor, community-mobilized events over 4 days. We estimated SARS-CoV-2 point prevalence (PCR positive) and cumulative incidence (antibody or PCR positive) in the census tract and evaluated risk factors for recent (PCR positive/antibody negative) vs prior infection (antibody positive/PCR negative). SARS-CoV-2 genome recovery and phylogenetics were used to measure viral strain diversity, establish viral lineages present, and estimate number of introductions.ResultsWe tested 3953 persons (40% Latinx; 41% White; 9% Asian/Pacific Islander; and 2% Black). Overall, 2.1% (83/3871) tested PCR positive: 95% were Latinx and 52% were asymptomatic when tested; 1.7% of census tract residents and 6.0% of workers (non-census tract residents) were PCR positive. Among 2598 tract residents, estimated point prevalence of PCR positives was 2.3% (95% confidence interval [CI], 1.2%-3.8%): 3.9% (95% CI, 2.0%-6.4%) among Latinx persons vs 0.2% (95% CI, .0-.4%) among non-Latinx persons. Estimated cumulative incidence among residents was 6.1% (95% CI, 4.0%-8.6%). Prior infections were 67% Latinx, 16% White, and 17% other ethnicities. Among recent infections, 96% were Latinx. Risk factors for recent infection were Latinx ethnicity, inability to shelter in place and maintain income, frontline service work, unemployment, and household income <$50 000/year. Five SARS-CoV-2 phylogenetic lineages were detected.ConclusionsSARS-CoV-2 infections from diverse lineages continued circulating among low-income, Latinx persons unable to work from home and maintain income during San Francisco's shelter-in-place ordinance