Biology subjects

Schellenberg, M. J.

Publications and source records attributed to Schellenberg, M. J..

4 recordsLinked to original sources

bioRxiv2026

SV40 Large T antigen inhibits the host serine protease FAM111A through a zinc-dependent, cleavage-avoiding mechanism

SV40 Large T antigen (LT) is essential for viral replication and a key determinant of host range. This host-range function is mediated by the C-terminal domain (LT-C) through binding to the host serine protease FAM111A, but the underlying mechanism has remained unclear. Here, we report the X-ray crystal structure of the FAM111A serine protease domain in complex with LT-C, revealing the structural basis for direct inhibition of FAM111A. LT-C uses a previously unrecognized zinc-binding motif and a P1-like phenylalanine residue to engage the FAM111A active site through a substrate-mimicking mechanism while avoiding proteolytic cleavage and covalent complex formation. Mutations disrupting either feature abolish FAM111A inhibition and impair SV40 propagation in cells. Consistent with this mechanism, SV40 host restriction requires FAM111A protease activity, which must be antagonized by LT-C for productive infection. Together, these findings define a zinc-dependent, cleavage-avoiding mechanism of protease inhibition that highlights an evolutionary arms race between SV40 and host antiviral proteases.

biochemistry↗

bioRxiv2021

High Efficiency Recombinant Protein Purification Using mCherry and YFP Nanobody Affinity Matrices

Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post-translational modifications in the proteins native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real-time monitoring of protein expression, and also provides an affinity handle for one-step protein purification using a suitable affinity reagent. Here we describe a panel of anti-GFP and anti-mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target protein using X-ray crystallography. From these analyses we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody-sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry-tag system by using it to purify recombinant human Topoisomerase 2 expressed in HEK293F cells. The mCherry-tag and GFP/YFP-tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real-time monitoring of protein expression levels and a high-efficiency purification step is needed.

biochemistry↗

bioRxiv2020

BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase II{beta} (TOP2B)-mediated DNA strand break and the activation of DNA damage response signaling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1 associated ring domain 1 (BARD1) complex, in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ATM and ATR during gene activation and that this event is essential for productive transcription. Our in vitro biochemical analyses showed TOP2B and BARD1 interaction and colocalization in the EGR1 transcription start site (TSS) and that the BRCA1-BARD1 complex ubiquitinates TOP2B, which appears to stabilize TOP2B protein in the cell and binding to DNA. Intriguingly, BRCA1 phosphorylation at S1524 controls this interaction. In addition, genomic analyses indicated colocalization between TOP2B and BRCA1 in a large number of protein-coding genes. Together, these findings reveal the novel function of the BRCA1-BARD1 complex in gene expression and in the regulation of TOP2B during Pol II transcription. Significance StatementMaintaining genomic integrity against cellular and extracellular genotoxic elements is essential for normal cell growth and function. Recent studies indicated that stimulus-induced transcription provokes topoisomerase II{beta}-mediated DNA strand break and DNA damage response signaling, requiring DNA repair to be coupled with transcription. Here, we present a novel role for the BRCA1-BARD1 complex in regulating the transcription of serum-inducible genes and the stability of topoisomerase II{beta}. The mechanism involving topoisomerase II{beta} ubiquitination by the BRCA1-BARD1 complex and the phosphorylation of BRCA1 S1524 upon transcriptional activation appears to function as a switch to the reaction. Our findings provide the first evidence of functional interaction between the BRCA1-BARD1 complex and topoisomerase II{beta} in transcription in humans.

molecular biology↗

bioRxiv2019

A genetic map of the response to DNA damage in human cells

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 28 CRISPR/Cas9 screens against 25 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 840 genes whose loss causes either sensitivity or resistance to DNA damaging agents. Mining this dataset, we uncovered that ERCC6L2, which is mutated in a bone-marrow failure syndrome, codes for a canonical non-homologous end-joining pathway factor; that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

cancer biology↗