Biology subjects

Lee, D. E.

Publications and source records attributed to Lee, D. E..

8 recordsLinked to original sources

bioRxiv2026

Allosteric Biosensors Unravel GTPase-Effector Feedback

Fluorescent biosensors that report protein conformation in vivo have been invaluable for understanding how the spatio-temporal dynamics of signaling controls cells. However, for GTPases these biosensors report the activated conformation using reagents that block the binding of downstream proteins, generating dominant negative effects and altering normal cell physiology. We present here a generalizable design to make GTPase biosensors (AlloRac1 and AlloCdc42), in which a circularly permuted fluorescent protein is inserted into a conserved loop allosterically connected to the effector binding site, generating activity-dependent fluorescence without blocking ligand interactions. The Rac1 biosensor showed that effector interactions led to increased Rac1 activation, indicating an auto-regulatory positive feedback made visible by the new biosensor design. This feedback regulated the kinetics and localization of Rac1 activity, including Rac1 activity gradients that controlled motility. Feedback was generated through Rac1 interaction with the effector Pak1, which led to further activation of Rac1 by the guanine exchange factor {beta}-Pix. The new biosensor approach enables quantitative imaging of previously obscure spatio-temporal dynamics in GTPase regulation.

cell biology↗

bioRxiv2024

Biochemical characterization of Bacillus anthracis sortase B: Use in sortase mediated ligation and substrate recognition dependent on residues beyond the canonical pentapeptide binding motif for sortase enzymes

Sortases are cysteine transpeptidases located on the surface of Gram-positive bacteria. These critical enzymes facilitate the attachment of proteins to the cell wall, and are potential targets for novel antibiotic development, as well as versatile tools in protein engineering applications. Although there are six classes of sortases recognized, class A sortases (SrtA) are the most widely studied and utilized. SrtA enzymes recognize the canonical Cell Wall Sorting Signal (CWSS), LPXTG, where X=any amino acid, although work in recent years identified additional promiscuity in multiple positions of this recognition motif. Much less is known about Class B sortases (SrtB), which target a distinct sequence, typically with an N-terminal Asn, e.g., variations of NPXTG or NPQTN. Although understudied overall, two SrtB enzymes have previously been shown to be specific for heme transporter proteins, and in vitro experiments with the catalytic domains of these enzymes reveal activities significantly worse than SrtA from the same organisms. Here, we use protein biochemistry, structural analyses, and computational simulations to better understand and characterize these enzymes, specifically investigating Bacillus anthracis SrtB (baSrtB) as a model SrtB protein. Structural modeling predicts a plausible enzyme-substrate complex, which is verified by mutagenesis of binding cleft residues at several positions. Furthermore, residues N- and C-terminal to the pentapeptide recognition motif are critical for observed activity. We also use chimeric proteins to identify a single site that improves baSrtB activity by [~]4-fold and use purified protein substrates to validate sortase-mediated ligation of two proteins using SrtB enzymes for the first time. Taken together, these studies provide insight into SrtB-target binding as well as evidence that SrtB enzymes can be modified to be of potential use in protein engineering.

biochemistry↗

bioRxiv2022

In-text citation error rate as a scientometric tool for evaluating accuracy and weighing evidence

Scientists are fallable and biased, but accuracy can be assessed through empirical analysis of published work that quantifies in-text citation (or quotation) errors. In scientific conflicts, it can be difficult for outsiders to know whose evidence or interpretation to trust. In-text citation error rate can assist decision- and policy-making bodies, as well as the courts when conflicts reach the judicial branch of government, by quantifying absolute and relative accuracy of scientists presenting scientific evidence. I propose the use of in-text citation error rates as a scientometric tool to quantify the accuracy of an authors work. In-text citation error rates in excess of an established overall mean (e.g., 11% for minor errors and 7% for major errors in ecology), or differences in in-text citation error rates between opposing groups of scientists could be used to reveal excessive inaccuracies in an author or group. The spotted owl (Strix occidentalis) has been at the center of a multi-decadal conflict caused by competition among people over forest resources, with scientific experts representing opposing stakeholders often presenting conflicting evidence. I applied the in-text citation error rate tool to important papers in the spotted owl and forest fire debate and found evidence of greater error rates in works on one side of this debate. In-text citation error rate can be an effective tool for quantifying accuracy among scientists.

scientific communication and education↗

bioRxiv2022

Phosphoproteomic mapping reveals distinct signaling actions and activation of protein synthesis and muscle hypertrophy by Isthmin-1

The secreted protein Isthmin-1 (Ism1) mitigates diabetes by increasing adipocyte and skeletal muscle glucose uptake by activating the PI3K-Akt pathway. However, while both Ism1 and insulin converge on these common targets, Ism1 has distinct cellular actions suggesting divergence in downstream intracellular signaling pathways. To understand the biological complexity of Ism1 signaling, we performed phosphoproteomic analysis after acute exposure, revealing overlapping and distinct pathways of Ism1 and insulin. We identify a 53 % overlap between Ism1 and insulin signaling and Ism1-mediated phosphoproteome-wide alterations in [~] 450 proteins that are not shared with insulin. Interestingly, we find several unknown phosphorylation sites on proteins related to protein translation, mTOR pathway and, unexpectedly, muscle function in the Ism1 signaling network. Physiologically, Ism1 ablation in mice results in altered proteostasis, including lower muscle protein levels under fed and fasted conditions, reduced amino acid incorporation into proteins, and reduced phosphorylation of the key protein synthesis effectors Akt and downstream mTORC1 targets. As metabolic disorders such as diabetes are associated with accelerated loss of skeletal muscle protein content, these studies define a non-canonical mechanism by which this anti-diabetic circulating protein controls muscle biology.

cell biology↗

bioRxiv2021

Multi-omic rejuvenation and lifespan extension upon exposure to youthful circulation

Heterochronic parabiosis (HPB) is known for its functional rejuvenation effects across several mouse tissues. However, its impact on the biological age of organisms and their long-term health remains unknown. Here, we performed extended (3-month) HPB, followed by a 2-month detachment period of anastomosed pairs. Old detached mice exhibited improved physiological parameters and lived longer than control isochronic mice. HPB drastically reduced the biological age of blood and liver based on epigenetic analyses across several clock models on two independent platforms; remarkably, this rejuvenation effect persisted even after 2 months of detachment. Transcriptomic and epigenomic profiles of anastomosed mice showed an intermediate phenotype between old and young, suggesting a comprehensive multi-omic rejuvenation effect. In addition, old HPB mice showed transcriptome changes opposite to aging, but akin to several lifespan-extending interventions. Altogether, we reveal that long-term HPB can decrease the biological age of mice, in part through long-lasting epigenetic and transcriptome remodeling, culminating in the extension of lifespan and healthspan.

cell biology↗

bioRxiv2019

Spreading depolarization and repolarization during cardiac arrest as an ultra-early marker of neurological recovery in a preclinical model

Spreading depolarization (SD) accompanies numerous neurological conditions, including migraine, stroke, and traumatic brain injury. There is significant interest in understanding the relationship between SD and neuronal injury. However, characteristics underlying SD and repolarization (RP) induced by global cerebral ischemia (e.g., cardiac arrest (CA)) and reperfusion are not well understood. Quantifying features of SD and RP during CA and cardiopulmonary resuscitation (CPR) may provide important metrics for diagnosis and prognosis of neurological injury from hypoxia-ischemia. We characterized SD and RP in a rodent model of asphyxial CA+CPR using a multimodal platform including electrocorticography (ECoG) and optical imaging. We detected SD and RP by (1) alternating current (AC), (2) direct current (DC), and (3) optical imaging of spreading ischemia, spreading edema, and vasoconstriction. Earlier SD (r=-0.80; p<0.001) and earlier RP (r=-0.71, p<0.001) were associated with better neurological recovery after 24hrs. SD+RP onset times predicted good vs poor neurological recovery with 82% sensitivity and 91% specificity. To our knowledge, this is the first preclinical study to link SD and RP characteristics with neurological recovery post-CA. These data suggest that SD and RP may be ultra-early, real-time prognostic markers of post-CA outcome, meriting further investigation into translational implications during global cerebral ischemia.

neuroscience↗

bioRxiv2019

Giraffe Translocation Population Viability Analysis

Most populations of giraffes have declined in recent decades, leading to the recent decision to upgrade the species to vulnerable status, and some subspecies to endangered. Translocations have been used as a conservation tool to re-introduce giraffes to previously occupied areas or establish new populations, but guidelines for founding populations are lacking. To provide general guidelines for translocation projects regarding feasibility, we simulated various scenarios of translocated giraffe populations to identify viable age and sex distributions of founding populations using Population Viability Analysis (PVA) implemented in Vortex software. We explored the parameter space for demography (population growth rates: {lambda} = 1.001, 1.010, 1.024), and the genetic load (number of lethal equivalents: LE = 2.5, 6.29, 12.6), examining how variation in founding numbers (N = 5 to 80 females) and sex ratios (M:F = 0.1 to 0.5) affected 100-year probability of extinction and genetic diversity. We found that even very small numbers of founders (N [≤]10 females) can appear to be successful in the first decades due to transient positive population growth, but with moderate population growth rate and moderate genetic load, long-term population viability (probability of extinction <0.01) was only achieved with [≥]30 females and [≥]3 males released. To maintain >95% genetic diversity of the source population in an isolated population, 50 females and 5 males are recommended to comprise the founding population. Sensitivity analyses revealed first-year survival and reproductive rate were the simulation parameters with the greatest proportional influence on probability of extinction and genetic diversity. These simulations highlight important considerations for translocation success, and data gaps including true genetic load in wild giraffe populations.

ecology↗

bioRxiv2017

Seeing spots: Measuring, quantifying heritability, and assessing fitness consequences of coat pattern traits in a wild population of giraffes (Giraffa camelopardalis)

Polymorphic phenotypes of mammalian coat color have been important to the study of genetics and evolution, but little is known about the heritability and fitness consequences of variation in complex coat pattern traits in wild populations. Understanding the current evolution of coat patterns requires reliably measuring traits, quantifying heritability of the traits, and identifying the fitness consequences of specific phenotypes. Giraffe coat markings are highly variable and it has been hypothesized that variation in coat patterns most likely affects fitness by camouflaging neonates against predators. We quantified spot pattern traits of wild Masai giraffes using image analysis software, determined whether spot pattern traits were heritable, and assessed whether variation in heritable spot pattern traits was related to fitness as measured by juvenile survival. The methods we described comprise a framework for objective quantification of mammalian coat pattern traits based on photographic coat pattern data. We demonstrated that characteristics of giraffe coat spot shape are heritable. We did not find evidence for juvenile survival consequences of variation in spot traits, suggesting that spot traits are currently not under strong directional, disruptive, or stabilizing selection for neonate camouflage in our study population, but our sample size could not detect small differences in survival. Spot trait variation also may be more relevant to other components of fitness, such as adult survival or fecundity. We hope this case study will inspire further investigations of coat pattern traits.

evolutionary biology↗