Background: Fowlpox virus (FPV) has been used as a vector for many years. To date, surprisingly, very little data exist on the persistence of fowlpox vaccines in chicken tissues, or what kind of immune cells respond to vaccination at the site of inoculation. Although both humoral and cellular- mediated immunity (CMI) play a part in overall immunity against FPV, little is known regarding the cell-mediated immune responses to FPV infection. Aim: The main aim of this paper was to measure persistence of fowlpox vaccine in skin tissues following vaccination. Methods: One-day-old chicks were vaccinated with Fowlpox virus FP9. Skin samples were collected from the site of inoculation for several days following vaccination to assess the presence of for FPV, as well as other immune cells including macrophages, CD4+ T cells, CD8+ T cells, and B cells. Results: Results showed that FPV does not persist for long, and is cleared by 6 days post-vaccination (dpv). The response of immune cells (macrophages, B cells, CD4+ and CD8+ T cells) that infiltrated the site of vaccination was assessed. Conclusion: The results in this paper reflected the response at the site of inoculation following single vaccination, therefore, further studies regarding viral persistence and infiltration of immune cells at the site of inoculation after booster vaccination should be done. [J Microbiol Infect Dis 2024; 14(3.000): 131-140]

Restricting the localization of evolutionarily conserved histone H3 variant CENP-A to the centromere is essential to prevent chromosomal instability (CIN), an important hallmark of cancers. Overexpressed CENP-A mislocalizes to non-centromeric regions and contributes to CIN in yeast, flies, and human cells. Centromeric localization of CENP-A is facilitated by the interaction of Mis18β with CENP-A specific chaperone HJURP. Cellular levels of Mis18β are regulated by β-transducin repeat containing protein (β-TrCP), an F-box protein of SCF (Skp1, Cullin, F-box) E3-ubiquitin ligase complex. Here, we show that defects in β-TrCP-mediated proteolysis of Mis18β contributes to the mislocalization of endogenous CENP-A and CIN in a triple-negative breast cancer (TNBC) cell line, MDA-MB-231. CENP-A mislocalization in β-TrCP depleted cells is dependent on high levels of Mis18β as depletion of Mis18β suppresses mislocalization of CENP-A in these cells. Consistent with these results, endogenous CENP-A is mislocalized in cells overexpressing Mis18β alone. In summary, our results show that β-TrCP-mediated degradation of Mis18β prevents mislocalization of CENP-A and CIN. We propose that deregulated expression of Mis18β may be one of the key mechanisms that contributes to chromosome segregation defects in cancers.

Mislocalization of overexpressed CENP-A (Cse4 in budding yeast, Cnp1 in fission yeast, CID in flies) contributes to chromosomal instability (CIN) in yeasts, flies, and human cells. Mislocalization of CENP-A is observed in many cancers and this correlates with poor prognosis. Structural mechanisms that contribute to mislocalization of CENP-A are poorly defined. Here, we show that interaction of histone H4 with Cse4 facilitates an in vivo conformational change in Cse4 promoting its mislocalization in budding yeast. We determined that Cse4 Y193A mutant exhibits reduced sumoylation, mislocalization, interaction with histone H4, and lethality in psh1Δ and cdc48-3 strains; all these phenotypes are suppressed by increased gene dosage of histone H4. We developed a new in vivo approach, antibody accessibility (AA) assay, to examine the conformation of Cse4. AA assay showed that wild-type Cse4 with histone H4 is in an ‘open’ state, while Cse4 Y193A predominantly exhibits a ‘closed’ state. Increased gene dosage of histone H4 contributes to a shift of Cse4 Y193A to an ‘open’ state with enhanced sumoylation and mislocalization. We provide molecular insights into how Cse4-H4 interaction changes the conformational state of Cse4 in vivo. These studies advance our understanding for mechanisms that promote mislocalization of CENP-A in human cancers.

p53 is a crucial tumor suppressor in vertebrates that is frequently mutated in human cancers. Most mutations are missense mutations that render p53 inactive in suppressing tumor initiation and progression. Developing small-molecule drugs to convert mutant p53 into an active, wild-type-like conformation is a significant focus for personalized cancer therapy. Prior research indicates that reactivating p53 suppresses cancer cell proliferation and tumor growth in animal models. Early clinical evidence with a compound selectively targeting p53 mutants with substitutions of tyrosine 220 suggests potential therapeutic benefits of reactivating p53 in patients. This study identifies and examines the UCI-1001 compound series as a potential corrector for several p53 mutations. The findings indicate that UCI-1001 treatment in p53 mutant cancer cell lines inhibits growth and reinstates wild-type p53 activities, including DNA binding, target gene activation, and induction of cell death. Cellular thermal shift assays, conformation-specific immunofluorescence staining, and differential scanning fluorometry suggest that UCI-1001 interacts with and alters the conformation of mutant p53 in cancer cells. These initial results identify pyrimidine trione derivatives of the UCI-1001 series as candidates for p53 corrector drug development.

Fowlpox virus (FPV) has been used as a vector for many years. To date, surprisingly, very little data exist on the persistence of fowlpox vaccines in chicken tissues, or what kind of immune cells respond to the vaccination at the site of inoculation. Although both humoral and cellular mediated immunity (CMI) play a part in overall immunity against FPV, little is known regarding the cell-mediated immune responses to FPV infection. The main aim of this paper was to measure persistence of the recombinant fowlpox vaccine in the skin tissues following vaccination. The results showed that the recombinant FPVs do not persist for long, and are cleared by 6 days post-vaccination (dpv). The response of immune cells (macrophages, B cells, CD4+ and CD8+ T cells) that infiltrating the site of vaccination was estimated.

Centromeric localization of evolutionarily conserved CENP-A (Cse4 in Saccharomyces cerevisiae) is essential for chromosomal stability. Mislocalization of overexpressed CENP-A to non-centromeric regions contributes to chromosomal instability (CIN) in yeasts, flies, and humans. Overexpression and mislocalization of CENP-A observed in many cancers is associated with poor prognosis. Previous studies have shown that F-box proteins, Cdc4 and Met30 of the Skp, Cullin, F-box (SCF) ubiquitin ligase cooperatively regulate proteolysis of Cse4 to prevent Cse4 mislocalization and CIN under normal physiological conditions. Mck1-mediated phosphorylation of SCF-Cdc4 substrates such as Cdc6 and Rcn1 enhances the interaction of the substrates with Cdc4. Here, we report that Mck1 interacts with Cse4, and Mck1-mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability. Our results showed that mck1Δ strain overexpressing CSE4 (GAL-CSE4) exhibits lethality, defects in ubiquitin-mediated proteolysis of Cse4, mislocalization of Cse4 and reduced Cse4-Cdc4 interaction. Strain expressing GAL-cse4-3A with mutations in three potential Mck1 phosphorylation consensus site (S10, S16, and T166) also exhibits growth defects, increased stability with mislocalization of Cse4-3A, CIN, and reduced interaction with Cdc4. Constitutive expression of histone H3 (Δ16H3) suppresses the CIN phenotype of GAL-cse4-3A strain, suggesting that the CIN phenotype is linked to Cse4-3A mislocalization. We conclude that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4-Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and CIN. These studies advance our understanding of pathways that regulate cellular levels of CENP-A to prevent mislocalization of CENP-A in human cancers.

AbstractThe F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.

Proteostasis fundamentally relies on the selective and timely turnover of thousands of intracellular proteins. In eukaryotes, the majority of this turnover is catalyzed by the ubiquitin-proteasome system (UPS), a vast array of enzymatic cascades that selectively target proteins for ubiquitylation followed by subsequent proteasomal degradation. Numerous regulatory mechanisms have evolved to modulate the UPS, which include the macromolecular machine known as the proteasome and other complex systems that covalently modify proteins with ubiquitin-like proteins. Dysregulation of the UPS has been associated with a myriad of human pathologies, and therapeutic strategies targeting proteasomal degradation have proven promising in clinical applications.

Abstract Dependence on exogenous methionine is a well-known, but poorly understood cancer-specific metabolic requirement. Although most cancer cells can convert homocysteine to methionine, they are unable to grow when methionine depleted media is supplemented with homocysteine. In contrast, non-tumorigenic cells proliferate well in homocysteine media. Previous work from our laboratory has shown that cancer cells cultured in homocysteine medium increase metabolic flux towards the transsulfuration pathway, away from S-adenosylmethionine (SAM) synthesis resulting in overall reduced methylation potential. We have shown that methionine sensing occurs independently of the mTOR pathway, which is known to link amino acid abundance and cell growth. Instead, our results suggest that Protein Phosphatase 2A (PP2A) methylation is sensitive to changes in the SAM/SAH ratio. Reduced PP2A methylation correlates with reduced cell proliferation in homocysteine medium. Furthermore, overexpression of the PP2A demethylase Pme1 forcibly reduces PP2A methylation and induces methionine dependence in cells that normally proliferate in homocysteine medium. These findings suggest a model that links methionine metabolism to cell cycle progression via regulation of PP2A methylation. Here we use quantitative proteomics to define changes in B-subunit binding to PP2A during methionine starvation to investigate the molecular mechanism of this methionine dependence seen in cancer. Citation Format: Anna Andronicos, Da-Wei Lin, Yi-Chen Su, Mari Ishak Gabra, Mei Kong, Peter Kaiser. Role of PP2A methylation on methionine dependence of cancer [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 6045.

In mammals, the role of interleukin-18 (IL-18) in the immune response is to drive inflammatory and, normally therefore, anti-viral responses. IL-18 also shows promise as a vaccine adjuvant in mammals. Chicken IL-18 (chIL-18) has been cloned. The aim of this study was to investigate the potential of chIL-18 to act as a vaccine adjuvant in the context of a live recombinant Fowlpox virus vaccine (fpIBD1) against Infectious bursal disease virus (IBDV). fpIBD1 protects against mortality, but not against damage to the bursa of Fabricius caused by IBDV infection. The Fowlpox virus genome itself contains several candidate immunomodulatory genes, including potential IL-18 binding proteins (IL-18bp). We knocked out (Δ) the potential IL-18bp genes in fpIBD1 and inserted (::) the cDNA encoding chIL-18 into fpIBD1 in the non-essential ORF030, generating five new viral constructs –fpIBD1::chIL-18, fpIBD1ΔORF073, fpIBD1ΔORF073::chIL-18, fpIBD1ΔORF214, and fpIBD1ΔORF214::chIL-18. The subsequent protection from challenge with virulent IBDV, as measured by viral load and bursal damage, given by these altered fpIBD1 strains, was compared to that given by the original fpIBD1. Complete protection was provided following challenge with IBDV in chicken groups vaccinated with either fpIBDIΔ073::IL-18 or fpIBD1Δ214::IL-18, as no bursal damage nor IBDV was detected in the bursae of the birds. The results show that chIL-18 can act as an effective vaccine adjuvant by improving the fpIBD1 vaccine and providing complete protection against IBDV challenge.

p53 is the main tumor suppressor protein in vertebrates and the most frequently mutated gene in human cancers. The majority of p53 mutations are missense mutations resulting in the expression of full-length p53 with inactive tumor suppressor function. Small molecule corrector drugs that change the mutant p53 conformation to a wild-type-like structure are attractive goals for personalized, targeted cancer therapies. Our previous work has shown that restoring p53 activities represses cancer cells and tumor growth in vitro and in vivo. This and other work suggest that reactivation of p53 could have therapeutic potential for cancer patients with mutated p53. This study aims to investigate a new potential p53 corrector small molecule, UCI-1001. Our data suggest that treatment of p53 mutant cancer cell lines with compounds of the UCI-1001 series inhibits cell growth in a p53 mutant-dependent manner and induces some p53-wild-type like activities such as mutant p53 DNA binding, transcriptional activation of p53 target genes, and induction of cell death. Utilizing the cellular thermal shift assay, CETSA, we show that UCI-1001 binds mutant p53 in vivo and changes mutant p53 conformation to a wild-type-like structure. Taken together, these preliminary results suggest the UCI-1001 series is a promising candidate for the development of p53 corrector drugs, but further development of pharmacokinetic properties will be necessary for translation to the bedside. Citation Format: Maryam Mohammed J Fallatah, Özlem Demir, Rommie Amaro, Peter Kaiser. A small molecule corrector for p53 mutants found in cancer [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 1716.

In mammals, the role of interleukin-18 (IL-18) in the immune response is to drive inflammatory and, normally therefore, anti-viral responses. IL-18 also shows promise as a vaccine adjuvant in mammals. Chicken IL-18 (chIL-18) has been cloned. The aim of this study was to investigate the potential of chIL-18 to act as a vaccine adjuvant in the context of a live recombinant Fowlpox virus vaccine (fpIBD1) against Infectious bursal disease virus (IBDV). fpIBD1 protects against mortality, but not against damage to the bursa of Fabricius caused by IBDV infection. The Fowlpox virus genome itself contains several candidate immunomodulatory genes, including potential IL-18 binding proteins (IL-18bp). We have knocked out (Δ) the potential IL-18bp genes in fpIBD1 and inserted (::) the cDNA encoding chIL-18 into fpIBD1 in the non-essential ORF030, generating five new viral constructs –fpIBD1::chIL-18, fpIBD1ΔORF073, fpIBD1ΔORF073::chIL-18, fpIBD1ΔORF214 and fpIBD1ΔORF214::chIL-18. The subsequent protection from challenge with virulent IBDV, as measured by viral load and bursal damage, given by these altered fpIBD1 strains, was compared to that given by the original fpIBD1. The results show that chIL-18 can act as a vaccine adjuvant, giving complete protection against challenge, with no detectable virus or damage in the bursa of Fabricius.

The tumor suppressor p53 is the most frequently mutated protein in human cancer and tops the list of high-value precision oncology targets. p53 prevents initiation and progression of cancer by inducing cell-cycle arrest and various forms of cell death. Tumors have thus evolved ways to inactivate p53, mainly by TP53 mutations or by hyperactive p53 degradation. This review focuses on two types of p53 targeting compounds, MDM2 antagonists and mutant p53 correctors. MDM2 inhibitors prevent p53 protein degradation, while correctors restore tumor suppressor activity of p53 mutants by enhancing thermodynamic stability. Herein we explore both novel and repurposed p53 targeting compounds, discuss their mode of action, and examine the challenges in advancing them to the clinic.

Colorectal cancer is among the most common cancers worldwide and a frequent cause of cancer related deaths. Oxaliplatin is the first line chemotherapeutics for treatment, but the development of resistance leads to recurrence of oxaliplatin insensitive tumors. To understand possible mechanisms of drug tolerance we developed oxaliplatin resistant derivatives (OR-LoVo) of the established LoVo cell line originally isolated from a metastatic colon adenocarcinoma. We compared the microRNA (miRNA) expression profile of the cell pair and found expression of miR-29a-3p significantly increased in OR-LoVo cells compared to parent cells. In addition, miR-29a-3p was significantly elevated in tumor tissue when compared to matched surrounding tissue in human, suggesting potential clinical importance. Ectopic miR-29-a-3p expression induced chemoresistance in a number of different cancer cell lines as well as colorectal tumors in mice. We further demonstrated that miR-29-a-3p downregulates expression of the ubiquitin ligase component FEM1B and that reduction of Fem1b levels is sufficient to confer oxaliplatin resistance. FEM1B targets the glioma associated oncogene Gli1 for degradation, suggesting that increased Gli1 levels could contribute to oxaliplatin tolerance. Accordingly, knockdown of GLI1 reverted chemoresistance of OR-LoVo cells. Mechanistically, resistant cells experienced significantly lower DNA damage upon oxaliplatin treatment, which can be partially explained by reduced oxaliplatin uptake and enhanced repair. These results suggest that miR-29-a-3p overexpression induces oxaliplatin resistance through misregulation of Fem1B and Gli1 levels. TCGA analyses provides strong evidence that the reported findings regarding induced drug tolerance by the miR-29a/Fem1B axis is clinically relevant. The reported findings can help to predict oxaliplatin sensitivity and resistance of colorectal tumors.

We successfully repurpose the DNA repair protein methylguanine methyltransferase (MGMT) as an inducible degron for protein fusions. MGMT is a suicide protein that removes alkyl groups from the O6 position of guanine (O6G) and is thereafter quickly degraded by the ubiquitin proteasome pathway (UPP). Starting with MGMT pseudosubstrates (benzylguanine and lomeguatrib), we first demonstrate that these lead to potent MGMT depletion while affecting little else in the proteome. We then show that fusion proteins of MGMT undergo rapid UPP-dependent degradation in response to pseudosubstrates. Mechanistic studies confirm the involvement of the UPP, while revealing that at least two E3 ligase classes can degrade MGMT depending on cell-line and expression type (native or ectopic). We also demonstrate the technique's versatility with two clinically relevant examples: degradation of KRASG12C and a chimeric antigen receptor.

Salmonella enterica serovar Enteritidis is a bacterial pathogen that contributes to poultry production losses and human foodborne illness. The bacterium elicits a broad immune response involving both the innate and adaptive components of the immune system. Coordination of the immune response is largely directed by cytokines. The objective of the current study was to characterize the expression of a select set of cytokines and regulatory immune genes in three genetically diverse chicken lines after infection with S. Enteritidis. Leghorn, Fayoumi and broiler day-old chicks were orally infected with pathogenic S. Enteritidis or culture medium. At 2 and 18 h postinfection, spleens and ceca were collected and mRNA expression levels for 7 genes (GM-CSF, IL2, IL15, TGF-β1, SOCS3, P20K, and MHC class IIβ) were evaluated by real-time quantitative PCR. Genetic line had a significant effect on mRNA expression levels of IL15, TGF-β1, SOCS3 and P20K in the spleen and on P20K and MHC class IIβ in the cecum. Comparing challenged vs. unchallenged birds, the expression of SOCS3 and P20K mRNA were significantly higher in the spleen and cecum, while MHC class IIβ mRNA was significantly lower in spleen. Combining the current RNA expression results with those of previously reported studies on the same samples reveals distinct RNA expression profiles among the three genetic chicken lines and the 2 tissues. This study illustrates that these diverse genetic lines have distinctively different immune response to S. Enteritidis challenge within the spleen and the cecum.

Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A to non-centromeric chromatin contributes to CIN in budding and fission yeasts, flies, and humans. Overexpression and mislocalization of CENP-A is observed in cancers and is associated with increased invasiveness. Mechanisms that remove mislocalized CENP-A and target it for degradation have not been defined. Here, we report that Cdc48 and its cofactors Ufd1 and Npl4 facilitate the removal of mislocalized Cse4 from non-centromeric chromatin. Defects in removal of mislocalized Cse4 contribute to lethality of overexpressed Cse4 in cdc48,ufd1 andnpl4 mutants. High levels of polyubiquitinated Cse4 and mislocalization of Cse4 are observed in cdc48-3, ufd1-2 and npl4-1mutants even under normal physiological conditions, thereby defining polyubiquitinated Cse4 as the substrate of the ubiquitin directed segregase Cdc48Ufd1/Npl4. Accordingly, Npl4, the ubiquitin binding receptor, associates with mislocalized Cse4, and this interaction is dependent on Psh1-mediated polyubiquitination of Cse4. In summary, we provide the first evidence for a mechanism that facilitates the removal of polyubiquitinated and mislocalized Cse4 from non-centromeric chromatin. Given the conservation of Cdc48Ufd1/Npl4 in humans, it is likely that defects in such pathways may contribute to CIN in human cancers.

Despite the successful control of highly contagious tumorigenic Marek’s disease (MD) by vaccination, a continuous increase in MD virus (MDV) virulence over recent decades has put emphasis on the development of more MD-resistant chickens. The cell types and genes involved in resistance therefore need to be recognized. The virus is primarily lymphotropic, but research should also focus on innate immunity, as innate immune cells are among the first to encounter MDV. Our previous study on MDV–macrophage interaction revealed significant differences between MHC-congenic lines 61 (MD-resistant) and 72 (MD-susceptible). To investigate the role of dendritic cells (DCs) in MD resistance, bone-marrow-derived DCs from these lines were infected with MDV in vitro. They were then characterized by cell sorting, and the respective transcriptomes analysed by RNA-seq. The differential expression (DE) of genes revealed a strong immune activation in DCs of the susceptible line, although an inherent immune supremacy was shown by the resistant line, including a significant expression of tumour-suppressor miRNA, gga-mir-124a, in line 61 control birds. Enrichment analysis of DE genes revealed high expression of an oncogenic transcription factor, AP-1, in the susceptible line following MDV challenge. This research highlights genes and pathways that may play a role in DCs in determining resistance or susceptibility to MDV infection.

Objective: The circadian clock aligns physiology with the 24-hour rotation of Earth. Light and food are the main environmental cues (zeitgebers) regulating circadian rhythms in mammals. Yet, little is known about the interaction between specific dietary components and light in coordinating circadian homeostasis. Herein, we focused on the role of essential amino acids. Methods: Mice were fed diets depleted of specific essential amino acids and their behavioral rhythms were monitored and tryptophan was selected for downstream analyses. The role of tryptophan metabolism in modulating circadian homeostasis was studied using isotope tracing as well as transcriptomic- and metabolomic- analyses. Results: Dietary tryptophan depletion alters behavioral rhythms in mice. Furthermore, tryptophan metabolism was shown to be regulated in a time- and light- dependent manner. A multi-omics approach and combinatory diet/light interventions demonstrated that tryptophan metabolism modulates temporal regulation of metabolism and transcription programs by buffering photic cues. Specifically, tryptophan metabolites regulate central circadian functions of the suprachiasmatic nucleus and the core clock machinery in the liver. Conclusions: Tryptophan metabolism is a modulator of circadian homeostasis by integrating environmental cues. Our findings propose tryptophan metabolism as a potential point for pharmacologic intervention to modulate phenotypes associated with disrupted circadian rhythms.