Biology subjects

King, V. L.

Publications and source records attributed to King, V. L..

3 recordsLinked to original sources

bioRxiv2026

Intracellular Delivery of Peptides and Proteins with an Engineered Membrane Translocation Domain

Antibodies and other protein therapeutics have revolutionized medicine, but their application is largely limited to extracellular targets. The lack of efficient intracellular delivery methods remains a major bottleneck. Here, we engineered a family of small ([~]90 amino acids), metabolically stable membrane translocation domains (MTDs) by modifying the loop sequences of a human fibronectin type III (FN3) domain. The most potent variant, MTD4, is highly cell-permeable and can be recombinantly fused to the N- or C-terminus of any peptide or protein, serving as a versatile "plug-and-play" vehicle. We demonstrate that MTD4 fusions efficiently deliver a wide variety of functional peptides and proteins into the cytosol and nucleus of eukaryotic cells, both in vitro and in vivo. Following systemic administration, MTD4 fusion proteins exhibit broad biodistribution and homogenous tissue penetration in mice. Importantly, MTD4 is effective at low nanomolar (nM) concentrations, making it a promising platform for addressing a vast range of intracellular and previously "undruggable" targets.

bioengineering↗

bioRxiv2022

F-actin-rich territories coordinate apoptosome assembly and caspase activation during intrinsic apoptosis

The actin cytoskeleton is a ubiquitous participant in cellular functions that maintain viability, but how it controls programmed cell death processes is not well understood. Here we show that in response to DNA damage, human cells form juxtanuclear F-actin-rich territories that coordinate the organized progression of apoptosome assembly to caspase activation. These cytoskeletal compartments are created by the actin nucleation factors JMY, WHAMM, and the Arp2/3 complex, and they exclude proteins that inhibit JMY and WHAMM activity. Within the territories, JMY localization overlaps with punctate structures containing the core apoptosome components cytochrome c and Apaf-1. The F-actin-rich areas also encompass initiator caspase-9 and clusters of a cleaved form of executioner caspase-3, while restricting accessibility of the caspase inhibitor XIAP. The clustering and potency of caspase-3 activation are positively regulated by the amount of actin polymerized by JMY and WHAMM. These results indicate that JMY-mediated actin reorganization functions in apoptotic signaling by coupling the biogenesis of apoptosomes to the localized processing of caspases.

cell biology↗

bioRxiv2020

The actin nucleation factors JMY and WHAMM enable a rapid p53-dependent pathway of apoptosis

The actin cytoskeleton is a well-known player in most vital cellular processes, but comparably little is understood about how the actin assembly machinery impacts programmed cell death pathways. In the current study, we explored roles for the human Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation factors in DNA damage-induced apoptosis. Inactivation of each WASP-family gene revealed that two, JMY and WHAMM, are required for rapid apoptotic responses. JMY and WHAMM enable p53-dependent cell death by enhancing mitochondrial permeabilization, initiator caspase cleavage, and executioner caspase activation. The loss of JMY additionally results in significant changes in gene expression, including upregulation of the small G-protein RhoD. Depletion or deletion of RHOD increases cell death, suggesting that RhoD normally plays a key role in cell survival. These results give rise to a model in which JMY and WHAMM promote intrinsic cell death responses that can be opposed by RhoD. Author SummaryThe actin cytoskeleton is a collection of protein polymers that assemble and disassemble within cells at specific times and locations. Cytoskeletal regulators called nucleation-promoting factors ensure that actin polymerizes when and where it is needed, and many of these factors are members of the Wiskott-Aldrich Syndrome Protein (WASP) family. Humans express 8 WASP-family proteins, but whether the different factors function in programmed cell death pathways is not well understood. In this study, we explored roles for each WASP-family member in apoptosis and found that a subfamily consisting of JMY and WHAMM are critical for a rapid pathway of cell death. Furthermore, the loss of JMY results in changes in gene expression, including a dramatic upregulation of the small G-protein RhoD, which appears to be crucial for cell survival. Collectively, our results point to the importance of JMY and WHAMM in driving intrinsic cell death responses plus a distinct function for RhoD in maintaining cell viability.

cancer biology↗