Biology subjects

Padrick, S. B.

Publications and source records attributed to Padrick, S. B..

4 recordsLinked to original sources

bioRxiv2022

Structure of VanS from Vancomycin-Resistant Enterococci: A Sensor Kinase with Weak ATP Binding

The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci (VRE). VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from VRE types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with KD values in the low millimolar range. Since these KD values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.

biochemistry↗

bioRxiv2022

A first-in-class Wiskott-Aldrich syndrome protein (WASp) activator with anti-tumor activity in hematological cancers

Hematological cancers are among the most common cancers in adults and in children. Despite significant improvements in therapies, many patients still succumb to the disease, therefore, novel therapies are needed. The Wiskott-Aldrich syndrome protein (WASp) family proteins regulate actin assembly in conjunction with the Arp2/3 complex, a ubiquitous nucleation factor. WASp is expressed exclusively in hematopoietic cells and exists in two allosteric conformations, auto-inhibited and active conformations. Here, we describe the development of EG-011, a first-in-class small molecule activator of the WASp auto-inhibited form. EG-011 possesses in vitro and in vivo anti-tumor activity as single agent in lymphoma, leukemia and multiple myeloma, including models of secondary resistance to PI3K, BTK and proteasome inhibitors. The in vitro activity was confirmed in a lymphoma xenograft. Actin polymerization induced by EG-011 was demonstrated with multiple techniques. Transcriptome analysis highlighted homology with drugs inducing actin polymerization. Key pointsO_LIEG-011 is a novel small molecule with anti-tumor activity in hematological cancers, including resistant lymphoma and multiple myeloma models C_LIO_LIEG-011 is a first-in-class small molecule activator of the auto-inhibited form of the Wiskott-Aldrich syndrome protein (WASp) C_LI

cancer biology↗

bioRxiv2022

Septins mediate a microtubule-actin crosstalk that enables actin growth on microtubules

Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons (1, 2). Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin (1, 2). Septins are filamentous GTP-binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin and intermediate filaments (3, 4). Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Super-resolution imaging shows that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We show that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings provide the first evidence of septins directly mediating microtubule interactions with actin filaments, and reveal a new mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis. Significance StatementCellular morphogenesis and processes such as cell division and cell migration require the coordination of the actin and microtubule cytoskeletons. Despite its broader physiological significance, actin-microtubule crosstalk is molecularly and mechanistically poorly understood. Actin-microtubule crosstalk has been viewed primarily as the regulation or guidance of microtubule dynamics by actin filaments. Here, we have discovered a new mechanism by which actin filament growth is guided by microtubules. We report that septins, a poorly understood component of the cytoskeleton, can capture and link the polymerizing ends of actin filaments to microtubule polymers. We present evidence that this new septin-mediated mechanism is critical for the morphology of growth cones, which are structures that direct the growth of neuronal axons through actin-microtubule crosstalk.

cell biology↗

bioRxiv2020

Production and analysis of a mammalian septin hetero-octamer complex

The septins are filament-forming proteins found in diverse eukaryotes from fungi to vertebrates, with roles in cytokinesis, shaping of membranes and modifying cytoskeletal organization. These GTPases assemble into rod-shaped soluble hetero-hexamers and hetero-octamers in mammals, which polymerize into filaments and higher order structures. While the cell biology and pathobiology of septins are advancing rapidly, mechanistic study of the mammalian septins is limited by a lack of recombinant hetero-octamer materials. We describe here the production and characterization of a recombinant mammalian septin hetero-octamer of defined stoichiometry, the SEPT2/SEPT6/SEPT7/SEPT3 complex. Using a fluorescent protein fusion to the complex, we observed filaments assembled from this complex. In addition, we used this novel tool to resolve recent questions regarding the organization of the soluble septin complex. Biochemical characterization of a SEPT3 truncation that disrupts SEPT3-SEPT3 interactions is consistent with SEPT3 occupying a central position in the complex while the SEPT2 subunits are at the ends of the rod-shaped octameric complexes. Consistent with SEPT2 being on the complex ends, we find that our purified SEPT2/SEPT6/SEPT7/SEPT3 hetero-octamer copolymerizes into mixed filaments with separately purified SEPT2/SEPT6/SEPT7 hetero-hexamer. We expect this new recombinant production approach to lay essential groundwork for future studies into mammalian septin mechanism and function.

biochemistry↗