About

Peter Brzovic is an Associate Professor of Biochemistry at the University of Washington. His research primarily investigates the structure and functions of macromolecular protein complexes involved in Ubiquitin signaling. He has focused on the central enzymes of Ubiquitin transfer pathways, specifically the E2 ubiquitin conjugating enzymes. His key research questions include the characterization of Ubiquitin-Conjugating Enzymes, understanding how they interact with E3 Ubiquitin ligases and substrates, and exploring how activated E2~Ub conjugates interact with effector proteins introduced into eukaryotic hosts from bacterial pathogens. Additionally, his work aims to understand the regulatory functions of E2 enzymes. His approaches primarily involve the use of NMR spectroscopy to understand the networks of interactions involving E2s and biochemical characterization of the complexes.

Research topics

• Biology
• Cell biology
• Biochemistry
• Genetics
• Chemistry
• Computational biology
• Biophysics
• Business
• Stereochemistry

Selected publications

• Abstract 6096: BRCA1-BARD1 ubiquitylates histones for genome maintenance

  Cancer Research · 2023-04-04

  article

  Abstract Background: Breast cancer type 1 susceptibility protein (BRCA1) is a tumor suppressor gene involved in DNA double strand break repair with well-known cancer implications. BRCA1 heterodimerizes with BRCA1 associated Ring domain 1 (BARD1) to form a complex with DNA binding and ubiquitin E3 ligase function capable of interacting with proteins of diverse biological processes, most notably homology-directed DNA repair. During DNA repair, BRCA1-BARD1 directly interfaces with nucleosomes and transfers mono ubiquitin (Ub) to lysine residues on the C-terminal tail of histone H2A. Although truncation of the enzymatic BRCA1-BARD1 RING-RING domain retains H2A ubiquitylating activity, full-length BRCA1-BARD1 binds more tightly with nucleosomes and displays higher H2A-Ub activity. However, the molecular basis and biological significance for this enhanced nucleosome binding and H2A-Ub activity is uncharacterized. Methods: Full length BRCA1-BARD1 or truncated mutants and histones were purified from E. coli. or insect cells. Nucleosomes were assembled for in vitro ubiquitylation reaction and binding assays. To determine the biological significance, mammalian cell lines that stably express wild type or mutant forms of BARD1 were established for cellular fractionation, foci analysis, and clonogenic survival studies alongside various DNA damage agents. Results: Our results show multiple interaction sites exist between BRCA1-BARD1 and nucleosomes which allow high-affinity chromatin binding and promote increased histone H2A ubiquitylation activity. Multivalent BARD1-nucleosome interactions, namely those using strong binding motifs located in the intrinsically disordered region (IDR) of BARD1, and the weak “kiss” interaction mediated by the RING domains of both BRCA1 and BARD1, are essential for H2A ubiquitylation by BRCA1-BARD1. Further, we isolated two types of specific histone binding and/or ubiquitylation-defective mutants of BARD1: a BARD1-IDR mutant with disrupted nucleosome binding withe retained H2A ubiquitylation ability, and a RING mutant that solely impairs H2A ubiquitylation. In both cases, we demonstrate that these mutants are hypersensitive to DNA damage agents, including polyADP-ribose polymerase (PARP) inhibitors, and demonstrate reduced capacity of BARD1 to associate with chromatin and foci formation owing to attenuated repair capacity. Conclusion: Our studies provide convincing evidence BRCA1-BARD1 interacts with nucleosomes and ubiquitylates histones via its E3 ligase activity. Further, it plays a critical role in DNA damage response and repair that contributes to genome stability, which when disrupted sensitizes them to DNA damage agents. Our results open new avenues towards understanding whether and how these mutations in BRCA1-BARD1 affect its tumor suppression functions and their implications clinically, ultimately with the goal to translate these findings for the benefit of cancer patients. Citation Format: Wenjing Li, Samuel R. Witus, Meiling Wang, Peter S. Brzovic, Rachel E. Klevit, Weixing Zhao. BRCA1-BARD1 ubiquitylates histones for genome maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6096.

  PublisherDOI

• Bacterial mimicry of eukaryotic HECT ubiquitin ligation

  bioRxiv (Cold Spring Harbor Laboratory) · 2023-06-05 · 1 citations

  preprintOpen access

  ABSTRACT HECT E3 ubiquitin (Ub) ligases direct their modified substrates toward a range of cellular fates dictated by the specific form of monomeric or polymeric Ub (polyUb) signal that is attached. How polyUb specificity is achieved has been a longstanding mystery, despite extensive study ranging from yeast to human. Two outlying examples of bacterial “HECT-like” (bHECT) E3 ligases have been reported in the human pathogens Enterohemorrhagic Escherichia coli and Salmonella Typhimurium, but what parallels can be drawn to eukaryotic HECT (eHECT) mechanism and specificity had not been explored. Here, we expanded the bHECT family and identified catalytically active, bona fide examples in both human and plant pathogens. By determining structures for three bHECT complexes in their primed, Ub-loaded states, we resolved key details of the full bHECT Ub ligation mechanism. One structure provided the first glimpse of a HECT E3 ligase in the act of ligating polyUb, yielding a means to rewire the polyUb specificity of both bHECT and eHECT ligases. Through studying this evolutionarily distinct bHECT family, we have not only gained insight into the function of key bacterial virulence factors but also revealed fundamental principles underlying HECT-type Ub ligation.

  PublisherOA PDFDOI

• Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair

  Molecular Cell · 2023-10-01 · 38 citations

  articleOpen access
  PublisherOA PDFDOI

• Bacterial ligases reveal fundamental principles of polyubiquitin specificity

  Molecular Cell · 2023-12-01 · 4 citations

  articleOpen access
  PublisherOA PDFDOI

• BRCA1/BARD1 intrinsically disordered regions facilitate chromatin recruitment and ubiquitylation

  The EMBO Journal · 2023-06-12 · 24 citations

  articleOpen accessCorresponding
  PublisherOA PDFDOI

• Antibacterial potency of Type VI amidase effector toxins is dependent on substrate topology and cellular context

  bioRxiv (Cold Spring Harbor Laboratory) · 2022 · 1 citations

  • Biology
  • Biochemistry
  • Computational biology

  Abstract Members of the bacterial T 6SS a midase e ffector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of n uclear m agnetic resonance (NMR) and a high-throughput in vivo genetic approach called d eep m utational s canning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

  PublisherOA PDFDOI

• Author response: Antibacterial potency of type VI amidase effector toxins is dependent on substrate topology and cellular context

  2022-06-23

  peer-reviewOpen access

  Distinct surfaces of an interbacterial competition cell wall toxin mediate interactions with different cellular binding partners, resulting in an inherent evolutionary trade-off across the toxin superfamily.

  PublisherDOI

• Author Reply to Peer Reviews of Antibacterial potency of Type VI amidase effector toxins is dependent on substrate topology and cellular context

  2022-04-26

  peer-review
  PublisherDOI

• BRCA1/BARD1 is a nucleosome reader and writer

  Trends in Biochemical Sciences · 2022 · 23 citations

  • Cell biology
  • Biology
  • Genetics
  PublisherOA PDFDOI

• BRCA1/BARD1 intrinsically disordered regions facilitate chromatin recruitment and ubiquitylation

  bioRxiv (Cold Spring Harbor Laboratory) · 2022-08-09 · 2 citations

  preprintOpen access

  Abstract BRCA1/BARD1 is a tumor suppressor E3 ubiquitin (Ub) ligase with roles in DNA damage repair and in transcriptional regulation. BRCA1/BARD1 RING domains interact with nucleosomes to facilitate mono-ubiquitylation of distinct residues on the C-terminal tail of histone H2A. These enzymatic domains constitute a small fraction of the heterodimer, raising the possibility of functional chromatin interactions involving other regions such as the BARD1 C-terminal domains that bind nucleosomes containing the DNA damage signal H2A K15-Ub and H4 K20me0, or portions of the expansive intrinsically disordered regions found in both subunits. Herein, we reveal novel interactions that support robust H2A ubiquitylation activity mediated through a high-affinity, intrinsically disordered DNA-binding region of BARD1. These interactions support BRCA1/BARD1 recruitment to chromatin and sites of DNA damage in cells and contribute to their survival. We also reveal distinct BRCA1/BARD1 complexes that depend on the presence of H2A K15-Ub, including a complex where a single BARD1 subunit spans adjacent nucleosome units. Our findings identify an extensive network of multivalent BARD1- nucleosome interactions that serve as a platform for BRCA1/BARD1-associated functions on chromatin.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R01GM09850305

  NIH · $283k · 2019

• NIH Grant R01GM098503

  NIH · $1.2M · 2019

• Defining the role of BARD1 in nucleosomal ubiquitylation

  NIH · $2.7M · 2021–2027

Frequent coauthors

• Rachel E. Klevit

  University of Washington

  48 shared

• Michael F. Dunn

  Universidad Nacional Autónoma de México

  16 shared

• Jonathan N. Pruneda

  Oregon Health & Science University

  9 shared

• Weixing Zhao

  8 shared

• Samuel R. Witus

  Howard Hughes Medical Institute

  7 shared

• Lea M. Starita

  7 shared

• Daniel Durocher

  Lunenfeld-Tanenbaum Research Institute

  7 shared

• Frank Sicheri

  Lunenfeld-Tanenbaum Research Institute

  6 shared

Education

• Ph. D., Biochemistry

  University of California, Riverside

  1991

• Bachelor of Arts, Chemistry

  University of California, San Diego

  1984