Cong, Anh

Abstract 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.

ABSTRACT Immediate early genes (IEGs) encode master transcription factors, including EGR1 , FOS , JUN , and MYC genes that drive robust transcription in the G1 phase of cell cycle. Our previous studies indicate that topoisomerase IIβ (TOP2B) critically regulates IEG transcription and that the activities of TOP2B are dynamically controlled through post-translational modifications by BRCA1-BARD1 and ERKs. Here, we show that two E2 enzymes, UBCH5b and UBC13/MMS2, differentiate the effects and functions of TOP2B ubiquitination by BRCA1-BARD1. Comprehensive transcriptomics, proteomics, and biochemical and molecular cellular analyses revealed a close relationship between BARD1 and key mitogen-activated protein kinase pathway genes and identified activated ERK2 as a novel kinase that phosphorylates BARD1 at S391, a previously reported mitotic phosphorylation site, whose genetic mutation has been linked to tumorigenesis. Mechanistically, the catalytic activity of ERK2 stimulates TOP2B ubiquitination mediated by BRCA1-BARD1 in complex with UBCH5b and UBC13/MMS2, which controls the binding and function of TOP2B and BARD1 for transcriptional activation at representative IEGs. Taken together, our data propose that there is a functional regulatory circuit involving TOP2B, BARD1, and ERK2, three key transcriptional activators for IEG transcription, in which the gene association and catalytic activity of TOP2B are regulated through E2-differentiated ubiquitination by BRCA1-BARD1 and the phosphorylation of BARD1 by ERK2 for productive transcription.

Anthracyclines are potent chemotherapeutic agents, yet their clinical use is hampered by their cardiotoxicity. Anthracyclines achieve potent anti- cancer effects primarily via TOP2A, but at the same time they induce a dose limiting cardiotoxicity through TOP2B. The use of a catalytic TOP2 inhibitors such as dexrazoxane (ICRF 187) significantly reduces the incidence of anthracycline-induced cardiotoxicity. However, dexrazoxane targets the highly conserved TOP2 dimer interface that is identical between TOP2A and TOP2B, fostering persisting concerns that its inhibition of TOP2A isoenzyme may interfere with anthracycline anticancer effects and augment anthracycline-induced myelosuppression. Thus, isoform- selective TOP2 inhibitors that can control the DNA damage caused by TOP2A vs TOP2B are a significant unmet clinical need. We have developed a new class of highly potent and TOP2B-selective catalytic inhibitors through rational design by building upon the general pharmacophore present in isolates from Calophyllum Brasiliense. Using X-ray crystallography, we demonstrate that our newly developed compounds occupy a previously unidentified druggable binding-site in the TOP2 ATPase domain and function as a physical barrier to block the conformational change triggered by ATP hydrolysis. We have named the class of inhibitors that target this site “obex”, the Latin word for obstacle. The obex binding site contains residues that differ between the TOP2A and TOP2B isoenzymes, based on which we rationally designed, prepared, and systematically evaluated a targeted panel of obex inhibitors. We have identified the lead molecule, namely, topobexin, as the first- in-class TOP2B-specific obex inhibitor that can prevent anthracycline-induced DNA damage. This first TOP2 isoform-specific inhibition underscores the broader potential to improve drug specificity and minimize adverse effects in various medical treatments.

Abstract The protein kinase C (PKC) family of enzymes are indispensable serine-threonine kinases involved in signal pathways implicated in many different cellular processes. Dysregulation of PKC isoforms may contribute to the development and progression of breast cancer. Z-Endoxifen (Z-ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM) exhibited potent antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancers. Mechanistically, we discovered that ENDX inhibits PKCβI and downstream AKT signaling. Based on insights from our recently determined crystal structure of PKCβI, we investigated the effects of ENDX on PKCβI in estrogen receptor-positive (ER+) breast cancer. In vitro FRET-based biochemical assays revealed that ENDX inhibits the kinase activity of conventional (PKCβI) as well as a novel (PKC;) PKC isoform with a similar IC50. However, the PKCβI catalytic domain was found to be less sensitive to ENDX compared to full-length PKCβI. We identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory model using X-ray crystallography. Since the spatial assembly of PKC is crucial for its kinase activity, we assessed the effects of ENDX on the intracellular localization of PKCβI using live cell confocal imaging. Sub-cellular tracking of YFP-tagged PKCβI in ER+ MCF7 cells paradoxically revealed that ENDX promoted the translocation of PKCβI to the plasma membrane in both a dose and time-dependent manner. Furthermore, co-treatment with inhibitors of PHLPP phosphatases, which regulate PKC, alleviated this translocation. Based on our prior data demonstrating greater antitumor activity of the Z vs E isomer of ENDX, we discovered that Z-ENDX inhibits PKCβI kinase activity more effectively than its E-isomer using in vitro kinase assays. Further, E-ENDX failed to promote PKCβI translocation to the membrane.Taken together, these results suggest that allosteric effects of Z-ENDX trigger PKCβI recruitment to the plasma membrane where it is normally active yet suppresses its kinase activity. These findings indicate that Z-ENDX induces a non-productive structure of the enzyme and "breaks" the well-known mechanism of PKC activation upon binding to the cell membrane. Moreover, Z-ENDX likely triggers PHLPP-mediated PKCβI dephosphorylation, and ultimately PKCβI degradation, suggesting that Z-ENDX represents a new mechanistic basis for targeting and downregulating PKCβI, and potentially other PKC family members, in cancer cells. This newly discovered function of Z-ENDX likely contributes to its superior anti-cancer effects when compared to TAM and other endocrine therapies. Citation Format: Sayantani Sarkar Bhattacharya, Huy V. Huynh, Jasmin K. Farmakes, Taylor L. Witter, Elizabeth S. Bruinsma, Swaathi Jayaraman, Anh T. Cong, John R. Hawse, Matthew P. Goetz, Matthew J. Schellenberg. Protein kinase C beta 1 (PKCβ1) is allosterically inhibited and paradoxically translocated to the membrane by z-endoxifen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB051.

Abstract Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation Sayantani Sarkar Bhattacharya1, Taylor L. Witter1, Anh Q. T. Cong1, Elizabeth Bruinsma2, Swaathi Jayaraman2, Matthew P. Goetz2, John R. Hawse1, and Matthew J. Schellenberg1 1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA 55905 2Department of Oncology, Mayo Clinic, Rochester, MN USA 55905 Background: Z-Endoxifen (ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM), exhibited high antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancer. ENDX has also been shown to be a protein kinase C (PKC) inhibitor. PKCs participate in diverse cellular functions and their activity is often elevated in breast tumors. Guided by mechanistic insights from our recently determined crystal structure of PKCβI, we sought to determine the effects of ENDX on PKCβI in a breast cancer cell line model. Methods: To determine how ENDX regulates PKC activity, we used a Z’LYTE kinase activity assay. This Fluorescence Resonance Energy Transfer (FRET) based biochemical method can detect differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. We probed changes in activity for conventional and novel PKCs, as well as the purified catalytic domain of conventional PKCs in vitro. Alongside, as kinase activity of PKC relies on its spatial assembly, therefore we studied its intracellular localization using live cell confocal imaging. MCF7 cells expressing YFP-tagged PKCβI were grown in a glass bottom chamber and treated with ENDX and other modulators for relevant time and doses for this study. Images were taken using Zeiss LSM 780 confocal laser scanning microscope and analyzed in Zeiss-ZEN microscope software and GraphPad Prism 9. Results: Our data from an in vitro kinase assay indicates that ENDX inhibits the kinase activity of conventional (PKCβI) and novel (PKC𝛿) PKC isoforms with a similar IC50, however PKCβI catalytic domain is less sensitive to ENDX. We also identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory mechanism. Our live cell imaging study demonstrated that ENDX promotes the recruitment of PKCβI to the cell membrane in both a dose and time-dependent manner. Moreover, this translocation can also be mitigated by co-treatment with inhibitors of the PKC-regulator PHLPP phosphatases. Conclusion: Taken together, these results suggest the allosteric effects of ENDX trigger PKCβI recruitment to the cell membrane, yet since ENDX also inhibits kinase activity it suggests that ENDX triggers a non-productive interaction with the enzyme and "breaks" the well-known mechanism of PKC activation upon binding the cell membrane. Furthermore, ENDX is likely to trigger dephosphorylation and ultimately degradation, suggesting that ENDX represents a new mechanistic basis for targeting and downregulating PKC in cancer cells. Hence, the current study provides an integrated pattern of highly specific treatments regimen that can exploit PKCβI as a repurposing clinical target in breast cancer. Citation Format: Sayantani Sarkar Bhattacharya, Taylor L. Witter, Anh T. Cong, Elizabeth S. Bruinsma, Swaathi Jayaraman, Matthew P. Goetz, John Hawse, Matthew Schellenberg. Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-03.

Abstract Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post‐translational modifications in the protein's 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 proteins 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 2α 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.

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA damage response signalling 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 ataxia-telangiectasia mutated and ATR during gene activation, and that this event is important for productive transcription. Our biochemical and genomic analyses showed that the BRCA1-BARD1 complex interacts with TOP2B in the EGR1 transcription start site and in a large number of protein-coding genes. Intriguingly, the BRCA1-BARD1 complex ubiquitinates TOP2B, which stabilizes TOP2B binding to DNA while BRCA1 phosphorylation at S1524 controls the TOP2B ubiquitination by the complex. Together, these findings suggest the novel function of the BRCA1-BARD1 complex in the regulation of TOP2B and Pol II-mediated gene expression.

Recent studies indicated that activated transcription of serum‐inducible genes in humans requires topoisomerase IIβ (TOP2B)‐mediated DNA strand break (DSB) and DNA damage response (DDR) signaling. However, the mechanisms by which TOP2B is regulated in this process are unknown. Here, we identified a novel role of BRCA1‐BARD1 complex in transcription of these genes by regulating TOP2B binding to the transcription start site (TSS). Our genomic and in vitro biochemical analyses demonstrated that BRCA1‐BARD1 complex and TOP2B colocalize in a large number of genes and physically interact for TOP2B ubiquitination. Upon transcriptional activation, BRCA1 is phosphorylated at S1524 by ataxia telangiectasia‐mutated (ATM) and ataxia telangiectasia and Rad3‐related protein (ATR). Intriguingly, unphosphorylatable BRCA1 (S1524A)‐BARD1 complex ubiquitinates TOP2B, but not WT or S1524D BRCA1. Using the TSS of a representative serum‐inducible gene, EGR1 , we found that ubiquitinated TOP2B binds to the DNA with much more stability than deubiquitinated one does. Together, our findings suggest that BRCA1‐BARD1 complex stabilizes TOP2B association with serum‐inducible genes through its E3 ligase function in transcription. Transcriptional activation induces TOP2B‐mediated DSB and DDR signaling to activate ATM/ATR that phosphorylates BRCA1 S1524. Phosphorylated BRCA1 is ineffective in ubiquitinating TOP2B, which loosens it from the DNA. We propose that this loosening may contribute to either mobility or removal of TOP2B.