Faculty Bibliography

Under osmotic stress, bacteria express a heterotetrameric protein complex, KdpFABC, which functions as an ATP-dependent K⁺ pump to maintain intracellular potassium levels. The subunit KdpA belongs to the superfamily of K⁺ transporters and adopts pseudo fourfold symmetry with a membrane-embedded selectivity filter as seen in K⁺ channels. KdpB belongs to the superfamily of P-type ATPases with a conserved binding site for ions within the membrane domain and three cytoplasmic domains that orchestrate ATP hydrolysis via an aspartyl phosphate intermediate. Previous work hypothesized that K⁺ moves parallel to the membrane plane through a 40 Å long tunnel that connects the selectivity filter of KdpA with a canonical binding site in KdpB. In the current work, we have reconstituted KdpFABC into lipid nanodiscs and used cryo-EM to image the wild-type pump under turnover conditions. We present a 2.1 Å structure of the E1~P·ADP conformation, which reveals new features of the conduction pathway. This map shows strong densities within the selectivity filter and at the canonical binding site, consistent with K⁺ bound at each of these sites in this conformation. Many water molecules occupy a vestibule and the proximal end of the tunnel, which becomes markedly hydrophobic and dewetted at the subunit interface. We go on to use ATPase and ion transport assays to assess effects of numerous mutations along this proposed conduction pathway. The results confirm that K⁺ ions pass through the tunnel and support the existence of a low-affinity site in KdpB for releasing these ions to the cytoplasm. Taken together, these data shed new light on the unique partnership between a transmembrane channel and an ATP-driven pump in maintaining the large electrochemical K⁺ gradient essential for bacterial survival.

Excessive accumulation of lipids within cardiomyocytes can sometimes initiate cardiomyopathy, while in other situations excess lipids do not cause harm. To understand how pathologic and non-pathologic lipid accumulation differ, we isolated lipid droplets (LDs) from two genetically altered mouse lines and from wild-type (WT) mice after an overnight fast. The LDs from MHC-peroxisomal proliferator-activated receptor γ1(MHC-Pparg1) transgenic mice were threefold larger than those from either fasted WT or non-cardiomyopathy MHC-diacylglycerol acyl transferase 1 (MHC-Dgat1) transgenic mice. Proteomic analysis of the LD-associated membrane proteins (LDAMPs) showed that MHC-Pparg1 LDs had less perilipin (PLIN). Proteins associated with lipolysis and LD formation (CIDEs and MTP), lipid synthesis, and Pparg signaling pathways were increased in MHC-Pparg1 LDAMPs. Unlike in MHC-Pparg1, MHC-Dgat1 LDAMPs exhibited increased mitochondrial peroxidative proteins with reduced adipose triglyceride lipase (Pnpla2), and Pparg coactivator 1 alpha (Pgc1A). Cardiomyocytes from MHC-Pparg1 hearts had transmission electron microscopy (TEM) images of ongoing lipolysis and greater amounts of lipolytic proteins. In contrast, images from MHC-Dgat1 cardiomyocytes showed more lipophagy. Consistent with the proteomic study and EM images, cardiac immunofluorescence staining showed that PLIN5 protein, thought to block LD lipolysis, was markedly reduced with MHC-Pparg1 overexpression, while hormone-sensitive lipase was increased. The autophagosome marker protein LC3B was increased in MHC-Dgat1 but not in MHC-Pparg1 hearts. Potentially toxic lipids like diacylglycerols and ceramides were increased in hearts but not LDs from MHC-Pparg1 mice. Our data indicate that cardiomyocyte LDs vary in size, composition, and metabolism. Cardiotoxicity was associated with greater LD lipolysis, which we postulate leads to intracellular release of toxic lipids.

Understanding the molecular mechanisms driving lineage decisions and differentiation during development is challenging in complex systems with a diverse progenitor pool, such as the mammalian cerebellum. Importantly, how different transcription factors cooperate to generate neural diversity and the gene regulatory mechanisms that drive neuron production, especially during the late stages of cerebellum development, are poorly understood. We used single cell RNA-sequencing (scRNA-seq) to investigate the developmental trajectories of Nestin-expressing progenitors (NEPs) in the neonatal mouse cerebellum. We identified FOXO1 as a key regulator of NEP-to-inhibitory neuron differentiation, acting directly downstream of ASCL1. Genome occupancy and functional experiments using primary NEP cultures showed that both ASCL1 and FOXO1 regulate neurogenesis genes during differentiation while independently regulating proliferation and survival, respectively. Furthermore, we demonstrated that WNT signalling promotes the transition from an ASCL1+ to a FOXO1+ cellular state. Finally, the role of WNT signalling in promoting neuron production via FOXO1 is conserved in primary human NEP cultures. By resolving how cerebellar inhibitory neurons differentiate, our findings could have implications for cerebellar disorders such as spinocerebellar ataxia, where these cells are overproduced.

Phosphorylation of hundreds of protein extracellular domains is mediated by two kinase families but the functional role of these kinases is underexplored. We find that the presynaptic release of the tyrosine-directed ectokinase, vertebrate lonesome kinase (VLK/Pkdcc), is necessary and sufficient for the direct extracellular interaction between EphB2 and GluN1 at synapses for phosphorylation of the ectodomain of EphB2 and mediation of injury-induced pain. Pkdcc is an essential gene in the nervous system, and VLK is enriched at synapses and released from neurons in an activity- and soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE)-dependent manner to drive extracellular interactions. Our results show that presynaptic sensory neuron-specific VLK knockout attenuates postsurgical pain in mice without changing sensorimotor performance, suggesting that VLK critically regulates synaptic protein-protein interactions and acute pain in response to injury.

INTRODUCTION/BACKGROUND:Tibial plateau fractures are some of the most commonly treated injuries around the knee and loss of range of motion has a significant effect on post-operative outcomes, very few studies have demonstrated the impact of flexion contractures. The purpose of this study was to determine the effect that development of a knee flexion contracture has on outcomes following operative repair of tibial plateau fractures. METHODS:Patients operatively treated for tibial plateau fractures (Schatzker II, IV, V, and VI) between 2005-2024 at a multi-center academic urban hospital system were included in this retrospective comparative study. Patients were grouped into 3 cohorts: 1. Full extension (FE), 2. 5-10 degrees of flexion contracture (Mild, ME) and 3. Greater than 10 degrees of flexion (Severe, SE) contracture at 6 months post-operatively. Patients with contracture were matched to patients who regained full extension based on age and Schatzker classification. Statistical analysis was used to evaluate outcomes including patient reported pain levels, Short Musculoskeletal Function Assessment (SMFA) scores, complication rates and reoperation rates. RESULTS:The cohort consisted of 3 groups of 30 patients (14 Schatzker II, 5 Schatzker IV, 3 Schatzker V, and 8 Schatzker VI). The average knee flexion contracture for the mild cohort was 5 degrees and the average knee flexion contracture for the severe cohort was 12.7 degrees. Patients who experienced flexion contracture had poorer SMFA scores at 6 months, and those in the severe cohort had the poorest SMFA scores (112.6) when compared to those with full extension at 6 months (77.7) (p<0.001). Flexion contractures were associated with higher rates of fracture related infection (FRI) (p =0.002). Patients with flexion contracture also had a higher rate of subsequent re-operation, with 36.7% of the ME undergoing re-operation and 40% of SE undergoing re-operation. CONCLUSIONS:Patients who developed a flexion contracture following repair of a tibial plateau fracture experienced worse outcomes, higher rates of complications, increased pain, and poorer function at long term follow up compared to those who achieved full knee extension.

BACKGROUND/UNASSIGNED:This study compares outcomes and complications of patients with Bado II Monteggia fracture-dislocations that required radial head fixation or replacement based upon approach to the radial head. METHODS/UNASSIGNED:A retrospective review was performed of 159 consecutive patients with proximal ulna fractures and a radial head dislocation or fracture (Monteggia Variant). Injuries were classified by Bado type. Forty-one patients with Bado II Monteggia injuries treated with either a radial head replacement or fixation with complete follow up were included. Demographics, injury information, surgical details, and follow up information including elbow range of motion (ROM) and complications were collected. A trans-osseous posterior (TOP) approach working through the ulna fracture to address the radial head first was used in 19 patients, while 22 patients had their radial head treated via a separate lateral (Kocher) interval after ulnar fixation. Ulnar plate fixation was performed for all patients. Comparisons were made using independent t-tests. RESULTS/UNASSIGNED:Forty-one Monteggia lesions treated through TOP (19, 46 %) or Kocher (22, 64 %) approaches underwent a radial head replacement (33, 80.5 %) or fracture repair (8, 19.5 %) with a mean final follow-up of 15.3 months. At all post-operative visits, groups displayed similar rates of functional elbow ROM. At latest follow-up rates of patient-reported pain, ultimate elbow ROM, time to radiographic healing were equivalent. No significant differences were observed in ulna non-union, joint malalignment, post-operative nerve injury, post-operative infection, heterotopic ossification, incidence of hardware failure, patient-reported pain, and rate of removal of symptomatic hardware. Sub-analysis of radial head replacement versus fixation revealed equivalent percentage of patients with full ROM at each post-operative time point. CONCLUSION/UNASSIGNED:For Bado II Monteggia fracture-dislocations, the surgical approach to the radial head-TOP versus Kocher-does not influence ultimate patient outcomes or complication rates. Radial head replacement and fixation provide comparable results. LEVEL OF EVIDENCE/UNASSIGNED:III.

Protein-protein interaction (PPI) networks are dynamically remodeled in disease, yet most systems biology approaches focus on changes in protein abundance, overlooking critical interaction-level dysfunction. Here, we present a robust, chemoproteomic method-dysfunctional Protein-Protein Interactome (dfPPI)-that enables high-throughput, systematic, disease-contextual mapping of PPI network dysfunctions in cells and primary human tissue. This method integrates chemical biology probes that selectively capture epichaperome-based interactome assemblies with label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and network-based computational analysis, to uncover the rewiring of protein networks not apparent from transcriptomic or proteomic data alone. The dfPPI platform can be applied across disease states, species, and tissues to identify actionable nodes of dysfunction and enable high-resolution, systems-level insights into disease progression. In this protocol, we demonstrate step-by-step procedures for sample preparation, chemical probe treatment, affinity enrichment, label-free LC-MS/MS analysis, and bioinformatics workflows used to generate and interpret dfPPI datasets. This article aims to promote reproducibility and accessibility of this approach, supporting its adoption by the broader systems biology and translational research communities.

Histone modifications play a key role in regulating gene expression and cell fate during development and disease. Current methods for cell-type-specific genome-wide profiling of histone modifications require dissociation and isolation of cells and are not compatible with all tissue types. Here we adapt Targeted DamID (TaDa) to recognize specific histone marks, by fusing chromatin-binding proteins or single-chain antibodies to Dam, an Escherichia coli DNA adenine methylase. When combined with TaDa, this enables cell-type-specific chromatin profiling in intact tissues or organisms. We first profiled H3K4me3, H3K9ac, H3K27me3 and H4K20me1 in vivo in neural stem cells of the developing Drosophila brain. Next, we mapped cell-type-specific H3K4me3, H3K9ac and H4K20me1 distributions in the developing mouse brain. Finally, we injected RNA encoding DamID constructs into 1-cell stage Xenopus embryos to profile H3K4me3 distribution during gastrulation and neurulation. These results illustrate the versatility of TaDa to profile cell-type-specific histone marks throughout the genome in diverse model systems.

BACKGROUND:Accumulated levels of mutant huntingtin protein (mHTT) and its fragments are considered contributors to the pathogenesis of Huntington's disease (HD). Stimulating autophagy may enhance clearance of mHTT and its aggregates which has been considered as a possible therapeutic strategy. However, the role and competence of the autophagy-lysosomal pathway (ALP) during HD progression in the human disease remains largely unknown. METHODS:Here, we used multiplex confocal and ultrastructural immunocytochemical analyses of ALP functional markers in relation to mHTT aggresome pathology in striatum and the less affected cortex or cerebellum of HD brains staged from Grade HD2 to HD4 by Vonsattel neuropathological criteria compared to controls. RESULTS:Immunolabeling revealed the localization of HTT/mHTT in ALP vesicular compartments labeled by autophagy-related adaptor proteins sequestosome 1 (p62/SQSTM1) and ubiquitin, and cathepsin D (CTSD) as well as HTT-positive inclusions. Although comparatively normal at HD2, neurons at later HD stages exhibited progressive enlargement and clustering of CTSD-immunoreactive autolysosomes/lysosomes and, ultrastructurally, autophagic vacuole/lipofuscin granules accumulated progressively, more prominently in striatum than cortex. These changes were accompanied by rises in levels of HTT/mHTT and p62/SQSTM1, particularly their fragments, in striatum but not in the cortex, and by increases of LAMP1 and LAMP2 RNA and LAMP1 protein. In addition, cargo-loaded autophagosomes and cathepsin-positive autolysosomes were readily observed, implying a lack of significant blockage in autophagosome formation and autophagosome-lysosome fusion. CONCLUSIONS:The findings collectively suggest that upregulated lysosomal biogenesis and preserved proteolysis maintain autophagic clearance in early-stage HD, but the observed progressive HTT build-up and AL accumulation at advanced disease stages may signify a failure in autophagy substrate clearance. These findings support the prospect that ALP stimulation applied at early disease stages, when clearance machinery is fully competent, could lead to therapeutic benefits in HD patients.