School of Medicine

Alpha B-crystallin (CryAB) is a small heat-shock protein highly expressed in cardiac tissue, where it functions as a molecular chaperone that helps prevent protein aggregation, particularly under stress conditions. A missense mutation in CryAB (R120G) causes autosomal dominant cardiomyopathy in humans and is characterized by extensive protein aggregation in cardiomyocytes. To better understand the pathogenic mechanisms underlying CryAB^R120G-associated cardiomyopathy, appropriate in vivo models are essential. Genetic mouse models are valuable tools for investigating disease pathogenesis and evaluating potential therapeutic strategies. In this study, we characterized a homozygous CryAB^R120G knock-in (KI) mouse model to assess the impact of this mutation on cardiac function. CryAB^R120G KI mice exhibited no overt changes in cardiac structure and function up to 12 months of age, with minimal changes in cardiac and proteotoxic stress markers, except for an increased atrial natriuretic peptide expression at 12 months. Protein quality control pathways remained largely unchanged. Although mitochondrial respiration was normal in young CryAB^R120G KI mice, it was reduced at 12 months of age. Despite the presence of insoluble protein aggregates, homozygous CryAB^R120G KI mice did not develop overt structural or functional cardiomyopathy through 12 months of age. These findings indicate that, within the age range examined, the CryAB^R120G KI model does not reproduce the overt cardiomyopathic phenotype associated with the CRYAB^R120G mutation in patients.

During cell stress, endoplasmic reticulum autophagy (ER-phagy) receptors remodel the ER by sequestering membrane proteins (cargo) into autophagosomes for degradation. The conserved ER-phagy receptor, Atg40, contains a motif that binds to Atg8 and a reticulon homology domain that is needed for vacuolar/lysosomal delivery. Cargo capture, however, requires the Atg40 binding partner Lst1/SEC24C. To address whether lipids regulate cargo capture during ER-phagy, we analyzed autophagy in neutral lipid-deficient cells. Unexpectedly, we found that Atg40 was delivered to the vacuole in autophagosomes without Lst1/SEC24C or cargo in mutant cells. Lipidomic analysis revealed changes in the ratio of phosphatidylethanolamine to phosphatidylcholine in the neutral lipid-deficient cells that are predicted to alter ER membrane bendability. Our findings imply that phospholipids control cargo sequestration by regulating receptor-cargo coupling at autophagic sites.

The structure of the catalytic subunit of cAMP-dependent protein kinase (PKA-C), a prototype for the protein kinase superfamily, laid the foundation for the development of targeted kinase inhibitors. Here we describe the structure and biophysical characterization of a PKA-C complex with BLU0588, a small PKA-selective inhibitor. The high-resolution crystal structure not only captures the inhibitor's unusual T-shaped geometry, but also shows how the four rings of BLU0588 serve as surrogates for ATP's adenosine and phosphate-organizing sites. Each site contains two subsites. BLU0588's planar azaindole and pyridine rings, which are buried beneath the glycine-rich loop in a hydrophobic shell at the base of the active site cleft, fill the adenine and ribose subsites. In contrast, BLU0588's indane and pyrrolidine rings fill the phosphate-organizing site. The indane ring occupies the α/β-phosphate organizing site while the pyrrolidine ring fills the Mg/γ-phosphate organizing site. The structure also shows how BLU0588 nucleates an open but stable conformation of the entire hydrophobic architecture of the N- and C-lobes. In addition to potently blocking phosphoryl transfer activity, BLU0588 also abolishes the synergistic high-affinity binding of the physiological pseudosubstrate inhibitor, protein kinase inhibitor. The residence time of BLU0588, measured by surface plasmon residence, contributes to its picomolar affinity and is distinct from H89, a commonly used but more promiscuous PKA inhibitor. These molecular insights provide a valuable framework for dissecting the organization of the active site cleft as well as different strategies for the rational design of more potent and selective kinase inhibitors in general.

Abstract Activating mutations in GNAQ and GNA11 (GNAQ oncogenes) are found in ∼93% of uveal melanoma (UVM) and 4% of skin cutaneous melanoma (SKCM), where they act as driver oncogenes. UVM is the most common primary cancer of the eye in adults, affecting more than 2,500 patients each year in the US alone, nearly 50% of whom will die from liver metastasis. To date, there are limited effective therapeutic options to prevent or treat UVM metastatic disease (mUVM), which typically also fails to respond to immunotherapies. By combining synthetic biology approaches, CRISPR/Cas9 genome-wide screens, and high-throughput chemogenetic drug screening, our team has revealed that classical and novel non-canonical GNAQ signaling circuits converge to promote UVM growth, survival, metastasis, and treatment resistance. Ultimately, elucidating GNAQ oncogenic signaling networks may reveal system vulnerabilities that can be exploited to develop new precision therapies for mUVM. In this regard, we have recently shown that darovasertib acts as a dual PKC-PKN inhibitor and exhibits the highest activity among thousands of drugs tested. Darovasertib has demonstrated encouraging activity in UVM patients, and clinical trials using darovasertib as a single agent in primary UVM lesions and in combination with crizotinib in mUVM are currently ongoing. However, few patients achieve complete responses, and tumors often progress due to the acquisition of resistance mechanisms. We aim to identify new targets that can overcome resistance to darovasertib. RNA-seq analysis revealed that long-term treatment with darovasertib increased the expression of YAP-target genes, and we hypothesized that YAP/TEAD activation may contribute to darovasertib resistance. Indeed, expression of an active YAP mutant (YAP2-5SA) or LATS1/2 inhibition was sufficient to induce darovasertib resistance in UVM cells. In turn, knockdown of YAP or TEAD, or the expression of a doxycycline-induced TEAD inhibitor (TEADi) peptide, increases darovasertib-induced apoptosis. Remarkably, co-targeting with small molecule TEADi decreases the expression of darovasertib-induced YAP targets and acts synergistically to reduce cell viability and increase UVM cell death. Ongoing studies are now exploring the preclinical benefit of combining darovasertib with small-molecule TEADi for the treatment of human UVM tumor xenografts in mice. Emerging evidence will be presented supporting that the Hippo YAP/TEAD pathway represents an adaptive mechanism of resistance to darovasertib treatment, and that the combination of TEADi with darovasertib may prevent the development of treatment resistance, thereby increasing the depth and duration of the anti-tumor response in UVM. Citation Format: Rodolfo Daniel Cervantes-Villagrana, Elena Sofia Cardenas Alcoser, Kuniaki Sato, Simone Lubrano, Tomohiko Ishikawa, Andrew E. Aplin, J. Silvio Gutkind. Targeting Hippo/YAP-TEAD increases the antitumor activity of darovasertib in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1837.

Cells release vesicles that serve important roles in long-range signaling and intercellular communication. These vesicles are released not just in response to stress, inflammation, injury, and chemoresistance, but also during... Cells release vesicles that serve important roles in long-range signaling and intercellular communication. These vesicles are released not just in response to stress, inflammation, injury, and chemoresistance, but also during homeostatic regulation. A particular class of vesicles called ectosomes or microparticles are released by the outward budding of the plasma membrane, a process which requires both the detachment of the membrane from the cortex and the exposure of negatively charged, curvature-inducing lipids such as phosphatidylserine from the inner leaflet to the outer leaflet. In this work, we develop a biophysical model that accounts for the interaction between these different factors. Using our model, we predict how linker properties influence outward budding of the plasma membrane and identify conditions that can promote or inhibit membrane curvature generation. These findings provide insight into the fundamental mechanisms underlying microparticle formation, elucidating the basic biology of this critical process. Further, these mechanistic insights may inspire techniques for inhibiting microparticles where they are harmful, such as chemoresistant drug efflux by tumor cells.

INTRODUCTION: Despite sepsis having growing awareness nationally, efforts to reduce the public health impact of sepsis have lagged. Although there are known pathophysiologic mechanisms and preventive strategies, sepsis is rarely approached as a predictable or preventable condition. Predicting who will develop sepsis in patients with infection still remains a challenge. This study examined modifiable and nonmodifiable risk factors associated with patients initially discharged home with an infection and had future sepsis-related admissions within 7 days of the index Emergency Department (ED) visit. METHODS: We conducted a multi-center retrospective cohort analysis of adults presenting to two university hospital EDs. The inclusion criteria encompassed adult patients who were discharged from the ED at their index visit with discharge diagnosis (ICD 10-CM code) of pneumonia, urinary tract infection (UTI), and/or cellulitis and who returned for hospital admission within 7 days of the index visit due to sepsis, severe sepsis without septic shock, and/or septic shock. Using multivariate regression, risk factors that predict return sepsis admission within 7 days of ED index visit were evaluated, and a 7-day return sepsis admission model was constructed. The predictive power of the model was measured by c-statistic. RESULTS: Among 10,179 unique ED patients, return sepsis admissions within 7 days occurred in 113 visits (1.11 % of discharged patients). Statistically significant risk factors among patients with infection associated with subsequent sepsis admission in the chosen model were Cardiovascular Disease (OR 2.07 95 % CI 1.26-3.42), Hypertension (OR 2.21 95 % CI 1.37-3.56), Chronic Kidney Disease (OR 1.80 95 % CI 1.11-2.91), Cancer (OR 2.22 95 % CI 1.43-3.45), Male (OR 1.67 95 % CI 1.13-2.45), arriving in an ambulance (vs. walk in OR 2.55 95 % CI 1.46-4.44), higher heart rate (OR 1.29 95 % CI 1.16-1.45), and higher temperature (OR 1.23 95 % CI 1.05-1.45), Hyperlipidemia was protective (OR 0.56 95 %CI 0.34-0.91). The c-statistic of our chosen model was 0.77 (95 % CI 0.73-0.81). The Hosmer-Lemeshow test for our logistic regression model resulted in a chi-square value of 7.23 with 8 degrees of freedom with a p-value of 0.51. This suggests that our model fits the data well. CONCLUSION: Our findings may be used to risk stratify and guide outpatient disposition decisions for ED patients with infection and to determine which patients need to be more closely monitored in the outpatient setting following ED discharge.

Programmes that support diversity, equity and inclusion (DEI) in science are under attack in the USA. Data indicate that diversity in the scientific workforce increases creativity and success in tackling challenging problems. Loss of promising talent supported by these programmes will substantially weaken our research capacity, limit innovation and substantially reduce discoveries important for driving scientific advancements.

Mitochondria are multifunctional organelles that are important for many different cellular processes, including energy production and biosynthesis of fatty acids, haem and iron–sulfur clusters. Mitochondrial dysfunction leads to a disruption in these processes, the generation of excessive reactive oxygen species, and the activation of inflammatory and cell death pathways. The consequences of mitochondrial dysfunction are particularly harmful in energy-demanding organs such as the heart. Loss of terminally differentiated cardiomyocytes leads to cardiac remodelling and a reduced ability to sustain contraction. Therefore, cardiomyocytes rely on multilayered mitochondrial quality control mechanisms to maintain a healthy population of mitochondria. Mitochondrial chaperones protect against protein misfolding and aggregation, and resident proteases eliminate damaged proteins through proteolysis. Irreparably damaged mitochondria can also be degraded through mitochondrial autophagy (mitophagy) or ejected from cells inside vesicles. The accumulation of dysfunctional mitochondria in cardiomyocytes is a hallmark of ageing and cardiovascular disease. This accumulation is driven by impaired mitochondrial quality control mechanisms and contributes to the development of heart failure. Therefore, there is a strong interest in developing therapies that directly target mitochondrial quality control in cardiomyocytes. In this Review, we discuss the current knowledge of the mechanisms involved in regulating mitochondrial quality in cardiomyocytes, how these pathways are altered with age and in disease, and the therapeutic potential of targeting mitochondrial quality control pathways in cardiovascular disease.

Here, we present a protocol for differentiating 3T3-L1 preadipocytes and stromal vascular fraction (SVF)-derived preadipocytes from mice into mature adipocytes, followed by the isolation of crude mitochondrial fractions. This cost-effective and reproducible protocol is optimized for small-plate formats, compatible with standard reagents, and suitable for metabolic studies such as insulin resistance and mitochondrial function.

The UBR family of ubiquitin ligases binds to N-termini of their targets (known as N-degron) to induce their ubiquitination and degradation via a conserved domain known as UBR-box. UBR1 and UBR2 share the highest sequence homology among the family, and substantial structural studies were previously performed for substrate binding by the UBR-boxes of UBR1 and UBR2. Here, we describe a new pocket in the UBR-boxes of UBR1 and UBR2 for binding the second residues of N-degrons through determining five co-crystal structures of the UBR-boxes with various N-degron peptides. Together with binding affinities measured by fluorescence polarization, we show that the two highly homologous UBR-boxes can interact with the second residue of an N-degron differently. In addition, the UBR-boxes undergo different conformational changes when binding N-degrons. Furthermore, we demonstrate that the sidechain of the third amino acid of an N-degron has no contribution to binding the UBR-boxes. These findings represent a new conceptual advancement for the UBR E3 ligases and the new insights described here can be leveraged for developing their selective ligands for research and potential therapies.