A Pathway to Solving the Structure of cl-Par-4 Tumor Suppressor Protein: Challenges & Findings

Prostate apoptosis response-4 (Par-4) is a pro-apoptotic tumor suppressor protein. Down-regulation of this protein has been reported in a myriad of cancers. Conversely, up-regulation of Par-4 is found to be associated with several neurodegenerative disorders. Par-4 is unique in the sense it can selectively induce apoptosis in cancer cells. For this, caspase-dependent intracellular cleavage of Par-4 is essential to produce the functionally active fragment, cl-Par-4 (caspase-cleaved Par-4). The cl-Par-4 protein inhibits the NF-κB-mediated cell survival pathway and causes selective apoptosis in various tumor cells. Our laboratory is interested in determining the structure of cl-Par-4 and understanding it’s interaction with various proteins. Currently, we are using biophysical techniques such as circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and SDS-PAGE to characterize the structure of cl-Par-4 in the presence of various concentrations of monovalent and divalent ions, in order to shed light on the possible ion-specific role of cl-Par-4 in inducing structure and self-association of this protein. Results show that effects on cl-Par-4 conformation are ion-specific, and effects of divalent cations are considerably more pronounced than effects from monovalent cations. We have also found that the cl-Par-4 shows a better stability in presence of Cl- ions than in presence of SO42- ions. Further, with the help of D313K mutant of cl-Par-4, we investigated that charge-charge repulsion between similar charged amino acid residues in leucine zipper is responsible for high salt at neutral pH or low salt at low pH requirement of cl-Par-4. All these findings will be helpful in getting the structured conformation of cl-Par-4 and, therefore, determining the structure of this protein via X-ray crystallography or via nuclear magnetic resonance (NMR).https://digitalcommons.odu.edu/gradposters2022_sciences/1013/thumbnail.jp

Characterization of Cl-Par-4: WT vs. Mutant

Intrinsically disordered proteins (IDPs) play important roles in regulation of cell signaling pathways as well as cellular processes. Dysregulation of these proteins is associated with several human diseases. Prostate apoptosis response-4 (Par-4), a proapoptotic tumor suppressor protein, is categorized as an intrinsically disordered protein and downregulation of this protein has been reported in myriad of cancers including glioma, breast cancers, and prostate cancers. The caspase-cleaved fragment of Par-4 (cl-Par-4) plays an active role in tumor suppression by inhibiting several cell survival pathways. Here, we employed site-directed mutagenesis to introduce a point mutation in the cl-Par-4 wildtype (WT) to generate the D313K cl-Par-4 mutant. We have characterized both the mutant and the WT using various biophysical techniques such as CD, DLS, and NMR to determine the effect of the D313K mutation. The results show that D313K cl-Par-4 attains a compact and well-folded helical conformation, possibly a tetramer, similar to that of the WT in presence of high salt at physiological pH. However, D313K does so with half the amount of salt required for the WT. This establishes that the substitution of a basic residue for an acidic residue at position 313 alleviates inter-helical charge repulsion between dimer partners and helps to stabilize the structural conformation.https://digitalcommons.odu.edu/gradposters2023_gradschool/1004/thumbnail.jp

Detection of viruses that have the potential to alter the pathogenicity of wild type Marek’s disease virus

Marek’s disease virus serotype one (MDV-1) causes neuropathic, cytolytic and lymphoproliferative disease in poultry. Chicken anaemia virus (CAV) is also a pathogen causing anaemia and immunosuppression in chicken. Marek’s disease serotype two virus (MDV-2) is naturally occurring in chicken and apathogenic virus. Natural multiple infection of these three viruses is possible. Presence of CAV and MDV-2 in the poultry flocks is potential to change in the pathogenicity of MDV-1 and also the pathogenicity of the CAV when infected alone.\ud \ud Three hundred and ten feather samples as three different panels were collected from representative broiler flocks throughout Australia. These broiler flocks had different\ud vaccination history against MDV. These samples were examined to detect the status of MDV-2 and CAV. The results of nested polymerase chain reaction (PCR) demonstrated that these two viruses are present in the broiler flocks. The samples were also investigated for MDV-1 by Mr. Ramon Layton (JCU) and the data from the MDV-1 investigation was analysed together with these two viruses. The flocks,\ud vaccinated with HVT (herpesvirus of turkey) were found to be free from both MDV-1 and MDV-2. However, after the cessation of vaccination, both MDV1 and MDV-2 reappeared, first MDV-1 and then MDV-2. Interestingly when the MDV-2\ud was increasing progressively, the MDV-1 was decreasing. Although the available data is not sufficient to draw a conclusion, it is strongly suggestive that the presence\ud of MDV-2 in infected birds changes the pathogenicity of MDV-1.The MDV-2 possibly works as a vaccine reducing the clinical MDV despite up to 30% prevalence of wild type MDV-1.\ud \ud The CAV was present in all the flocks tested with more or less similar pattern of the viral distribution ranged between 50%-100%. Two sets of nested primers were used\ud for detection of the CAV variably reacted with samples. The difference in the reactivity of the primer was then assumed as primer sequence mismatch due to CAV strain differences. This project further aimed to determine the source of CAV in the broilers. The CAV can be transmitted through both horizontal and vertical route. Both parents can transmit the virus to the progeny. To control clinical disease in\ud young chicks, breeding flocks are generally vaccinated with a live vaccine.\ud \ud Possible source of CAV infection in the broiler flocks and the genetic divergence of the CAV detected in the samples were determined by sequencing the CAV field isolates selected on the basis of reactivity differences of two nested primer pairs and geographical location of the broiler farms. Eight field isolates, vaccine strain 3711\ud and a reference strain BF4 were sequenced generating 951 bp long sequences covering complete VP3 gene, 3’ 86% of VP2 gene and starting 42% of VP1 gene including hypervariable region. The sequence alignment and evolutionary analysis of\ud these data, only Australian isolates and along with other global CAV isolates demonstrated that the Australian CAV field isolates fitted in to three different groups. Vaccine (strain 3711), strain BF4 and one of the field isolates included in a group, six field isolates included in next group together with CIA-1, and remaining one field isolate included in the third group together with the Cux-1.\ud \ud Although one of the field isolate is included in the group along with vaccines, the isolate had genetic differences sufficient to differentiate from the strain 3711. These\ud results demonstrated that the CAV in the broilers is not the vaccine virus. In addition this group of virus have two non-synonymous mutations in the VP3 gene, not described to dates in the sequences of CAV global isolates except in the sequences of strain CAU/7 (an Australian isolate).\ud \ud To differentiate possible vertical and horizontal transmission of CAV in the broiler flocks, weekly sequential samples from week one till slaughter from one of the previous test flock was collected and investigated by nested PCR. The nested PCR result demonstrated that the birds were infected from week one. Eighty percent of the\ud samples tested contained CAV DNA in it. This result further indicated that the virus was transmitted vertically. However the infection was sustained to the flock with\ud similar pattern of distribution in the subsequent week samples till slaughter.\ud \ud Nearly full length genomic identification of the vaccine virus was determined (19 bp shorter). In attempts to sequence full length genome of one field isolates from three\ud genetically different groups of isolates, five different sequence data were obtained. Three overlapping primer pairs were designed to complete the sequences of the remaining 1347/1368 bp long DNA. The same primers were used for amplification of vaccine strain as well. Two sequences, 723 bp and 438 bp long were identified as chicken genome, one 563 bp long as Psychrobacter arcticus 273-4 and two\ud sequences, 1.28 kb and 499 bp long do not have sequence identification more than twenty base pair except primer sequence as CAV at both end of the sequence. These\ud sequence data demonstrated that chicken/other bacteria also have the primer sequence similarity in their genome enough to initiate DNA amplification in that given condition. In addition, these sequences data of chicken genomes or would be chicken genome or the bacterial genome further indicates reliability of the single round PCR results if used as diagnostic tool.\ud \ud The results from this project demonstrated that MDV-2 and CAV are present in Australian broilers. The presence of MDV-2 infection in the broiler flock prior to MDV-1 infection is likely to decrease the pathogenicity of MDV-1. Every broiler flocks is likely to have CAV infection. At lease three different strains of CAV are circulating in the present Australian commercial broilers. The CAV strains were not the vaccine virus administered to the parental flocks. The CAV strains were wild type virus most likely vertically transmitted to the broilers and they were maintained in the broiler shed environment

Enhancing the Conformational Stability of the cl-Par-4 Tumor Suppressor via Site-Directed Mutagenesis

Intrinsically disordered proteins play important roles in cell signaling, and dysregulation of these proteins is associated with several diseases. Prostate apoptosis response-4 (Par-4), an approximately 40 kilodalton proapoptotic tumor suppressor, is a predominantly intrinsically disordered protein whose downregulation has been observed in various cancers. The caspase-cleaved fragment of Par-4 (cl-Par-4) is active and plays a role in tumor suppression by inhibiting cell survival pathways. Here, we employed site-directed mutagenesis to create a cl-Par-4 point mutant (D313K). The expressed and purified D313K protein was characterized using biophysical techniques, and the results were compared to that of the wild-type (WT). We have previously demonstrated that WT cl-Par-4 attains a stable, compact, and helical conformation in the presence of a high level of salt at physiological pH. Here, we show that the D313K protein attains a similar conformation as the WT in the presence of salt, but at an approximately two times lower salt concentration. This establishes that the substitution of a basic residue for an acidic residue at position 313 alleviates inter-helical charge repulsion between dimer partners and helps to stabilize the structural conformation