Scripps (United States)

Abstract Extracellular targeted protein degradation (eTPD) has expanded access to the “undruggable” proteome but is constrained by aspects of receptor-mediated endocytosis and intracellular trafficking. This study describes Shed dase- Ta rgeting C himeras ( SHEDTACs ), bispecific antibodies that commandeer endogenous membrane metalloproteases (sheddases) to catalyze direct proteolysis of cell surface targets in cis . SHEDTACs bypass internalization requirements through enforced proximity between sheddases and substrates, enabling rapid proteolysis directly at the cell surface. Induced proximity between the immune checkpoint LAG3 and metalloprotease ADAM10 afforded nearly quantitative receptor depletion from primary human T cells. LAG3 shedding was rapid and unaffected by pharmacologic perturbation of proteasomal or lysosomal pathways, consistent with cell surface proteolysis. In a luminescent T cell reporter co-culture, SHEDTACs catalyzed LAG3 removal and enhanced T cell receptor signaling beyond conventional blocking antibodies, addressing resistance mechanisms in cancer immunotherapy. Notably, SHEDTACs depleted the non-canonical sheddase substrate, PD-1, demonstrating this approach can be expanded beyond natural protease-target pairs. SHEDTACs establish a mechanistically orthogonal eTPD platform that exploits cell surface proteolysis, offering programmable control over the cell surface proteome with broad therapeutic implications.

Catalytic reactions involving multiple cycles are common in many key organic transformations. We report a detailed mechanistic analysis of one such network of "nested" cycles, the Ni-catalyzed cross-coupling of alkyl sulfonyl hydrazides with (hetero)aryl halides, employing a battery of techniques, including Temperature Scanning Reaction (TSR) calorimetry coupled with Reaction Progress Kinetic Analysis (RPKA), quantitative NMR time-course analysis, kinetic modeling, linear free energy analysis, and density functional theory (DFT) quantum chemical calculations. TSR/RPKA studies yield the simultaneous determination of the thermodynamic enthalpy of the reaction, the kinetic rate law, and the activation parameters (ΔH‡, ΔS‡, ΔG‡), for the rate-determining step. Expanding the analysis to the coupling of an array of electron-poor and electron-rich sulfonyl hydrazides (each readily accessible on the decagram scale) with a range of (hetero)aryl halide coupling partners introduces the concept of "kinetics matching," and demonstrates that optimal yields may be obtained when the reactivities of the separate cycles are tuned to work in unison. These requirements for reaction networks involving nested catalytic cycles are showcased by several real-world examples. Our work highlights the predictive power of this kinetics-focused mechanistic approach, which may be extended to other nested catalytic networks to select appropriate catalyst and substrate combinations for optimal outcomes.

Enterococcus faecium is a member of the human gut microbiota that has evolved into a problematic nosocomial pathogen and a leading cause of infections in hospitalized patients. Treatment of E. faecium infections is complicated by antibiotic resistance, making it important to understand resistance mechanisms and their broader consequences in this pathogen. Here, we explored the collateral effects of rifampin resistance-associated mutations in the E. faecium RNA polymerase β-subunit (RpoB). Of 14,384 publicly available E. faecium genomes, nearly one-third carried a mutation in the rifampin resistance-determining region (RRDR) of RpoB. In a local population of 710 E. faecium clinical isolates collected from patients at a single hospital, we found significant associations between the presence of RRDR mutations and prior exposure to rifamycin antibiotics, as well as associations between RRDR mutations and altered daptomycin susceptibility. To investigate the phenotypic impacts of RRDR mutations, we generated and studied four isogenic strains with distinct RRDR mutations (Q473K, G482D, H486Y, and S491L) that overlapped with clinical isolate variants. Transcriptomic and phenotypic analyses revealed allele-specific effects on E. faecium gene expression, growth dynamics, antibiotic susceptibility, isopropanol tolerance, and cell wall physiology. One frequently observed mutation, H486Y, caused minimal transcriptional changes and enhanced bacterial fitness under antibiotic stress. In contrast, the S491L mutation induced extensive transcriptional changes and slowed bacterial growth but also conferred increased isopropanol tolerance, potentially enhancing bacterial survival on hospital surfaces. Overall, our findings highlight the multifaceted impacts of RRDR mutations in shaping E. faecium physiology and antibiotic resistance, two important features of this hospital-associated pathogen.IMPORTANCEUnderstanding how antimicrobial resistance affects bacterial physiology is critical for developing effective therapeutics against bacterial infections. In this study, we found that rifampin resistance-associated mutations in RpoB are widespread in Enterococcus faecium, a leading multidrug-resistant pathogen. By studying isogenic wild-type and RpoB mutant strains, we discovered that RpoB mutations, although conferring resistance to rifampin, have distinct allele-specific effects on other bacterial phenotypes. Some of these collateral effects appear to promote E. faecium resistance to antibiotics and survival in the hospital environment, raising questions about the selective pressures driving their emergence. Overall, our study underscores the importance of examining the collateral effects of resistance-associated mutations in multidrug-resistant pathogens, which could help mitigate their persistence and spread among vulnerable patients.

Abstract Informal caregiving continues to increase, and caregivers are becoming more diverse, prompting continued efforts to examine the impact of informal caregiving. Focusing on caregivers in the higher education landscape is beginning to gain more traction as the impact of population aging and caregiver diversity becomes more evident. Supporting faculty and staff caregivers in higher education is essential for fostering a more inclusive and equitable environment, allowing them to more effectively balance professional and caregiving responsibilities. The evidence-based age-inclusivity domains of higher education (AIDHE) model guides institutions of higher education in assessing age-inclusive practices that impact faculty and staff across seven domains of institutional function. Data from a campus-wide caregiving study revealed that nearly one in four employees (24.6%) identified as caregivers, most commonly to aging parents and spouses. Stress from caregiving, work conflicts, and care for others in the household were cited as limiting caregiving ability. Approximately half (47.5%) experienced low quality of life due to lacking caregiver support. The majority (58.5%) were unaware of support services for caregiving. The experiences of faculty and staff caregivers highlight the need for better recognition and support services from institutions of higher education. Based on study findings and framed through the lens of the AIDHE model, recommendations will be made for universities to capitalize on pre-existing campus programs and resources, and to develop new age-inclusive programs and practices to effectively respond to the needs of faculty and staff caregivers.

Extrachromosomal DNA (ecDNA) is a prevalent and devastating form of oncogene amplification in cancer1,2. Circular megabase-sized ecDNAs lack centromeres, stochastically segregate during cell division3–6 and persist over many generations. It has been more than 40 years since ecDNAs were first observed to hitchhike on mitotic chromosomes into daughter cell nuclei, but the mechanism underlying this process remains unclear3,7. Here we identify a family of human genomic elements, termed retention elements, that tether episomes to mitotic chromosomes to increase ecDNA transmission to daughter cells. Using Retain-seq, a genome-scale assay that we developed, we reveal thousands of human retention elements that confer generational persistence to heterologous episomes. Retention elements comprise a select set of CpG-rich gene promoters and act additively. Live-cell imaging and chromosome conformation capture show that retention elements physically interact with mitotic chromosomes at regions that are mitotically bookmarked by transcription factors and chromatin proteins. This activity intermolecularly recapitulates promoter–enhancer interactions. Multiple retention elements are co-amplified with oncogenes on individual ecDNAs in human cancers and shape their sizes and structures. CpG-rich retention elements are focally hypomethylated. Targeted cytosine methylation abrogates retention activity and leads to ecDNA loss, which suggests that methylation-sensitive interactions modulate episomal DNA retention. These results highlight the DNA elements and regulatory logic of mitotic ecDNA retention. Amplifications of retention elements promote the maintenance of oncogenic ecDNA across generations of cancer cells, and reveal the principles of episome immortality intrinsic to the human genome.

Live-cell mapping reveals ligandable RNA sites and guides design of selective RNA degraders.

KIF5A interacts with mRNA encoding synaptic ribosomal proteinsKIF5A is required for normal synaptic composition and functionKIF5A binds to G3BP1 and G3BP1 stress granule associated proteinsKIF5A mutant ALS patient-derived motor neurons have abnormal synaptic function and stress response.

Abstract Description MAIT cells are functionally diverse innate-like lymphocytes that recognize microbial-derived antigens. While MAIT cells can exhibit both tissue repair and cytotoxic properties, their role in chronic inflammatory skin disease is unknown. Using MAIT cell specific tetramer, we found that MAIT cells are reduced in skin of mice that develop chronic inflammatory skin disease, mimicking human cutaneous lupus erythematosus (CLE). Like human lupus skin, MAIT cell levels are ∼10-fold lower in pre-lesional skin of young MRL-lpr lupus-prone mice, compared to control MRL-MpJ or C57Bl/6 skin. MAIT cells in CLE-like skin have an activated/exhausted phenotype. Topical dosing of MAIT cell antigen, 5-OP-RU, expands MAIT cells in MRL-lpr skin, decreases expression of inflammatory (Il6, Tnf) and cytotoxic (Gzmb, Ifng) genes, and stimulates local Treg expansion. Treg do not expand with topical 5-OP-RU in MAIT-deficient, confirming Treg expansion is MAIT cell dependent. Of therapeutic relevance, short-term expansion of skin MAIT cells results in complete and lasting resolution of severe skin lesions in MRL-lpr mice, while vehicle-treated mice never fully heal. These data suggest that the absence of MAIT cells may contribute to pathogenesis of chronic skin inflammation and that MAIT cell-driven immunosuppression in the skin may be mediated via Treg expansion. Overall, these findings have implications for skin disease in systemic LE (SLE), one of the leading causes of death in young females. Funding Sources T32 AI007363 NIAMS R01AR080641 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

Small molecule ligands targeting structured RNA elementshold promisefor modulating RNA function, serving as chemical probes and potentialtherapeutics. In this study, the characterization of phenylglyoxal-basedcovalent probe designed to target unpaired guanine residues in structuredRNAs is reported. A structure-guided design strategy was employedto modify covalently unpaired guanines critical for flavin mononucleotide(FMN) binding to the FMN riboswitch. Covalent modification occursat the designed site and modulates riboswitch function in a cellularreporter system, highlighting the potential of covalent mechanismsof action for bioactive RNA ligands.