Priyadarshan K. Damle

Staphylococcal pathogenicity island interference with helper phage reproduction is a paradigm of molecular parasitism

Staphylococcal pathogenicity islands (SaPIs) carry superantigen and resistance genes and are extremely widespread in Staphylococcus aureus and in other Gram-positive bacteria. SaPIs represent a major source of intrageneric horizontal gene transfer and a stealth conduit for intergeneric gene transfer; they are phage satellites that exploit the life cycle of their temperate helper phages with elegant precision to enable their rapid replication and promiscuous spread. SaPIs also interfere with helper phage reproduction, blocking plaque formation, sharply reducing burst size and enhancing the survival of host cells following phage infection. Here, we show that SaPIs use several different strategies for phage interference, presumably the result of convergent evolution. One strategy, not described previously in the bacteriophage microcosm, involves a SaPI-encoded protein that directly and specifically interferes with phage DNA packaging by blocking the phage terminase small subunit. Another strategy involves interference with phage reproduction by diversion of the vast majority of virion proteins to the formation of SaPI-specific small infectious particles. Several SaPIs use both of these strategies, and at least one uses neither but possesses a third. Our studies illuminate a key feature of the evolutionary strategy of these mobile genetic elements, in addition to their carriage of important genes—interference with helper phage reproduction, which could ensure their transferability and long-term persistence.

Capsid size determination by Staphylococcus aureus pathogenicity island SaPI1 involves specific incorporation of SaPI1 proteins into procapsids

The Staphylococcus aureus pathogenicity island SaPI1 carries the gene for the toxic shock syndrome toxin (TSST-1) and can be mobilized by infection with S. aureus helper phage 80alpha. SaPI1 depends on the helper phage for excision, replication and genome packaging. The SaPI1-transducing particles comprise proteins encoded by the helper phage, but have a smaller capsid commensurate with the smaller size of the SaPI1 genome. Previous studies identified only 80alpha-encoded proteins in mature SaPI1 virions, implying that the presumptive SaPI1 capsid size determination function(s) must act transiently during capsid assembly or maturation. In this study, 80alpha and SaPI1 procapsids were produced by induction of phage mutants lacking functional 80alpha or SaPI1 small terminase subunits. By cryo-electron microscopy, these procapsids were found to have a round shape and an internal scaffolding core. Mass spectrometry was used to identify all 80alpha-encoded structural proteins in 80alpha and SaPI1 procapsids, including several that had not previously been found in the mature capsids. In addition, SaPI1 procapsids contained at least one SaPI1-encoded protein that has been implicated genetically in capsid size determination. Mass spectrometry on full-length phage proteins showed that the major capsid protein and the scaffolding protein are N-terminally processed in both 80alpha and SaPI1 procapsids.

The roles of SaPI1 proteins gp7 (CpmA) and gp6 (CpmB) in capsid size determination and helper phage interference.

SaPIs are molecular pirates that exploit helper bacteriophages for their own high frequency mobilization. One striking feature of helper exploitation by SaPIs is redirection of the phage capsid assembly pathway to produce smaller phage-like particles with T=4 icosahedral symmetry rather than T=7 bacteriophage capsids. Small capsids can accommodate the SaPI genome but not that of the helper phage, leading to interference with helper propagation. Previous studies identified two proteins encoded by the prototype element SaPI1, gp6 and gp7, in SaPI1 procapsids but not in mature SaPI1 particles. Dimers of gp6 form an internal scaffold, aiding fidelity of small capsid assembly. Here we show that both SaPI1 gp6 (CpmB) and gp7 (CpmA) are necessary and sufficient to direct small capsid formation. Surprisingly, failure to form small capsids did not restore wild-type levels of helper phage growth, suggesting an additional role for these SaPI1 proteins in phage interference.

A conformational switch involved in maturation of Staphylococcus aureus bacteriophage 80α capsids.

Bacteriophages are involved in many aspects of the spread and establishment of virulence factors in Staphylococcus aureus, including the mobilization of genetic elements known as S. aureus pathogenicity islands (SaPIs), which carry genes for superantigen toxins and other virulence factors. SaPIs are packaged into phage-like transducing particles using proteins supplied by the helper phage. We have used cryo-electron microscopy and icosahedral reconstruction to determine the structures of the procapsid and the mature capsid of 80α, a bacteriophage that can mobilize several different SaPIs. The 80α capsid has T=7 icosahedral symmetry with the capsid protein organized into pentameric and hexameric clusters that interact via prominent trimeric densities. The 80α capsid protein was modeled based on the capsid protein fold of bacteriophage HK97 and fitted into the 80α reconstructions. The models show that the trivalent interactions are mediated primarily by a 22-residue β hairpin structure called the P loop that is not found in HK97. Capsid expansion is associated with a conformational switch in the spine helix that is propagated throughout the subunit, unlike the domain rotation mechanism in phage HK97 or P22.

Constitutive Activation of DNA Damage Checkpoint Signaling Contributes to Mutant p53 Accumulation via Modulation of p53 Ubiquitination

Many mutant p53 proteins exhibit an abnormally long half-life and overall increased abundance compared with wild-type p53 in tumors, contributing to mutant p53s gain-of-function oncogenic properties. Here, a novel mechanism is revealed for the maintenance of mutant p53 abundance in cancer that is dependent on DNA damage checkpoint activation. High-level mutant p53 expression in lung cancer cells was associated with preferential p53 monoubiquitination versus polyubiquitination, suggesting a role for the ubiquitin/proteasome system in regulation of mutant p53 abundance in cancer cells. Interestingly, mutant p53 ubiquitination status was regulated by ataxia–telangectasia mutated (ATM) activation and downstream phosphorylation of mutant p53 (serine 15), both in resting and in genotoxin-treated lung cancer cells. Specifically, either inhibition of ATM with caffeine or mutation of p53 (serine 15 to alanine) restored MDM2-dependent polyubiquitination of otherwise monoubiquitinated mutant p53. Caffeine treatment rescued MDM2-dependent proteasome degradation of mutant p53 in cells exhibiting active DNA damage signaling, and ATM knockdown phenocopied the caffeine effect. Importantly, in cells analyzed individually by flow cytometry, p53 levels were highest in cells exhibiting the greatest levels of DNA damage response, and interference with DNA damage signaling preferentially decreased the relative percentage of cells in a population with the highest levels of mutant p53. These data demonstrate that active DNA damage signaling contributes to high levels of mutant p53 via modulation of ubiquitin/proteasome activity toward p53. Implication: The ability of DNA damage checkpoint signaling to mediate accumulation of mutant p53 suggests that targeting this signaling pathway may provide therapeutic gain. Mol Cancer Res; 14(5); 423–36. ©2016 AACR.

An intestinal stem cell niche in Apc mutated neoplasia targetable by CtBP inhibition

C-terminal binding protein 2 (CtBP2) drives intestinal polyposis in the Apcmin mouse model of human Familial Adenomatous Polyposis. As CtBP2 is targetable by an inhibitor of its dehydrogenase domain, understanding CtBP2s role in adenoma formation is necessary to optimize CtBP-targeted therapies in Apc mutated human neoplasia. Tumor initiating cell (TIC) populations were substantially decreased in ApcminCtbp2+/- intestinal epithelia. Moreover, normally nuclear Ctbp2 was mislocalized to the cytoplasm of intestinal crypt stem cells in Ctbp2+/- mice, both Apcmin and wildtype, correlating with low/absent CD133 expression in those cells, and possibly explaining the lower burden of polyps in Apcmin Ctbp2+/- mice. The CtBP inhibitor 4-chloro-hydroxyimino phenylpyruvate (4-Cl-HIPP) also robustly downregulated TIC populations and significantly decreased intestinal polyposis in Apcmin mice. We have therefore demonstrated a critical link between polyposis, intestinal TICs and Ctbp2 gene dosage or activity, supporting continued efforts targeting CtBP in the treatment or prevention of Apc mutated neoplasia.

Abstract 2571: DBC1, a novel CBP-interacting protein, promotes p53 stability by regulating CBP-dependent p53 polyubiquitination

The acetyltransferase CBP, in conjunction with the E3 ubiquitin ligase enzyme, Mdm2, maintains physiologic levels of the tumor suppressor protein p53 in the absence of cellular stress, via a cytoplasmic, but not nuclear, p53-directed E4 polyubiquitin activity. CBP also possesses cytoplasmic, but not nuclear, E3 ubiquitin ligase, autoubiquitination activity. To understand the mechanism by which the ubiquitin ligase activities of CBP are compartmentalized in the cell, we have employed Multidimensional Protein Identification Technology (MudPIT) to identify cytoplasmic and nuclear CBP interacting proteins. MudPIT analysis revealed that Cell Cycle and Apoptosis Regulator protein (CCAR2), also known as Deleted in Breast Cancer 1 protein (DBC1), is a novel CBP- interacting protein in both the nucleus and cytoplasm. The N-terminus of DBC1 bound to multiple CBP domains, including the putative E3/E4 domain of CBP located at the N-terminus. Interaction between DBC1 and CBP suppressed the in vitro E3 autoubiquitination activity of CBP. Functional studies demonstrated that DBC1 directly regulates the cellular compartmentalization of CBP E3 and p53-directed E4 ubiquitin ligase activities. Knockdown of DBC1 in U2OS cells stimulated normally absent nuclear p53 polyubiquitination, and also caused a decrease in p53-dependent and p53-independent apoptosis, rescued by CBP/DBC1 double knockdown. Further, over-expression of DBC1 rescued the increase in nuclear p53 polyubiquitination and decrease in p53 half-life observed in DBC1 depleted cells. Together, these results identify DBC1 as a novel binding partner of CBP and a critical regulator of CBP ubiquitin ligase activities. Based on our data that DBC1 loss inactivates p53 function through destabilization, we queried the TCGA database for co-occurrence of DBC1 loss with maintenance of wild-type p53 status which showed that 33% of breast, 40% of lung, and 96% of prostate cancer cases with DBC1 alterations maintained wild-type p53 status. Therefore, by restoring DBC1 function, this work could lead to reactivation of p53 tumor suppressor activity in tumors with DBC1 alterations maintaining wild-type p53 status. Citation Format: Oluwatoyin E. Akande, Priyadarshan K. Damle, Nicholas E. Sherman, Steven R. Grossman. DBC1, a novel CBP-interacting protein, promotes p53 stability by regulating CBP-dependent p53 polyubiquitination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2571. doi:10.1158/1538-7445.AM2017-2571

Abstract 2199: Evaluation of critical residues in the C-terminal binding protein (CtBP) dehydrogenase domain contributing to substrate binding, catalysis, and oncogenic activity

C-terminal binding proteins (CtBP) 1 and 2 are transcriptional coregulators that are upregulated in several cancers, including a majority of studied breast, colorectal, and ovarian tumor samples. CtBPs drive many cellular oncogenic properties, including migration, invasion, proliferation, and survival, in part through repression of tumor suppressor genes, such as E-cadherin, PTEN, BRCA1, and p16. CtBPs encode an intrinsic dehydrogenase activity regulated by both intracellular NADH concentration and the putative substrate 4-methylthio-2-oxobutyric acid (MTOB). NADH binding induces CtBP dimerization, which regulates the recruitment of transcriptional regulatory complexes. High levels of MTOB appear to inhibit CtBP dehydrogenase function and induce cytotoxicity among cancer cells in a CtBP-dependent manner. However, the function of the substrate-binding domain has yet to be examined with regard to CtBP9s oncogenic activity. To this end, we have created several point mutations in the substrate-binding pocket of CtBP and have determined key residues for CtBP9s enzymatic activity using an in vitro dehydrogenase assay. We have found that a conserved tryptophan in the catalytic domain, and specifically its aromatic moiety, is imperative for enzyme activity. This tryptophan is unique to CtBP family proteins and distinguishes the CtBP substrate-binding domain from that of all other families of dehydrogenases. Additionally, we found arginine and histidine residues lining the substrate pocket that are necessary for CtBP dehydrogenase function. Knowledge of these residues allows the directed synthesis of drugs with increased potency and higher CtBP specificity. Moreover, to investigate the importance of the catalytic domain on CtBP9s oncogenic potential, we have transfected human cancer cell lines with several CtBP substrate-binding domain mutants and observed the effects on cellular processes, including growth, survival, migration, and recruitment of key components of the transcriptional repressor complex. From this work we have investigated the utility of CtBP, and specifically the CtBP substrate-binding domain, as a target for cancer therapeutics. We also provide preliminary insights into the function of this domain in cellular models of cancer. Citation Format: Benjamin L. Morris, Priyadarshan Damle, Zaid Nawaz, Steven R. Grossman. Evaluation of critical residues in the C-terminal binding protein (CtBP) dehydrogenase domain contributing to substrate binding, catalysis, and oncogenic activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2199. doi:10.1158/1538-7445.AM2015-2199

Molecular Piracy via Capsid Size Determination by Staphylococcus aureus Pathogenicity Island 1

* Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294 ** Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294 *** Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294 **** Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298