Proteomics Analysis of the Polyomavirus DNA Replication Initiation Complex Reveals Novel Functional Phosphorylated Residues and Associated Proteins.

Polyomavirus (PyV) Large T-antigen (LT) is the major viral regulatory protein that targets numerous cellular pathways for cellular transformation and viral replication. LT directly recruits the cellular replication factors involved in initiation of viral DNA replication through mutual interactions between LT, DNA polymerase alpha-primase (Polprim), and single-stranded DNA binding complex, (RPA). Activities and interactions of these complexes are known to be modulated by post-translational modifications; however, high-sensitivity proteomic analyses of the PTMs and proteins associated have been lacking. High-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of the immunoprecipitated factors (IPMS) identified 479 novel phosphorylated amino acid residues (PAARs) on the three factors; the function of one has been validated. IPMS revealed 374, 453, and 183 novel proteins associated with the three, respectively. A significant transcription-related process network identified by Gene Ontology (GO) enrichment analysis was unique to LT. Although unidentified by IPMS, the ETS protooncogene 1, transcription factor (ETS1) was significantly overconnected to our dataset indicating its involvement in PyV processes. This result was validated by demonstrating that ETS1 coimmunoprecipitates with LT. Identification of a novel PAAR that regulates PyV replication and LT's association with the protooncogenic Ets1 transcription factor demonstrates the value of these results for studies in PyV biology.

Proteomics analysis reveals novel phosphorylated residues and associated proteins of the polyomavirus DNA replication initiation complex.

Polyomavirus ( PyV ) Large T-antigen ( LT ) is the major viral regulatory protein that targets numerous cellular factors/pathways: tumor suppressors, cell cycle regulators, transcription and chromatin regulators, as well as other factors for viral replication. LT directly recruits the cellular replication factors involved in LT's recognition of the viral origin, origin unwinding, and primer synthesis which is carried out by mutual interactions between LT, DNA polymerase alpha-primase ( Polprim ), and single strand (ss) DNA binding replication protein A ( RPA ). The activities as well as interactions of these three with each other as well as other factors, are known to be modulated by post-translational modifications (PTMs); however, modern high-sensitivity proteomic analyses of the PTMs as well as proteins associated with the three have been lacking. Elution from immunoprecipitation (IP) of the three factors were subjected to high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified 479 novel phosphorylated amino acid residues (PAARs) on the three factors: 82 PAARs on SV40 LT, 305 on the Polprim heterotetrametric complex and 92 on the RPA heterotrimeric complex. LC-MS/MS analysis also identified proteins that co-immunoprecipitated (coIP-ed) with the three factors that were not previously reported: 374 with LT, 453 with Polprim and 183 with RPA. We used a bioinformatic-based approach to analyze the proteomics data and demonstrate a highly significant "enrichment" of transcription-related process associated uniquely with LT, consistent with its role as a transcriptional regulator, as opposed to Polprim and RPA associated proteins which showed no such enrichment. The most significant cell cycle related network was regulated by ETS proto-oncogene 1 (ETS1), indicating its involvement in regulatory control of DNA replication, repair, and metabolism. The interaction between LT and ETS1 is validated and shown to be independent of nucleic acids. One of the novel phosphorylated aa residues detected on LT from this study, has been demonstrated by us to affect DNA replication activities of SV40 Large T-antigen. Our data provide substantial additional novel information on PAARs, and proteins associated with PyV LT, and the cellular Polprim-, RPA- complexes which will benefit research in DNA replication, transformation, transcription, and other viral and host cellular processes.

A fluorescence-based, gain-of-signal, live cell system to evaluate SARS-CoV-2 main protease inhibition.

The likelihood of continued circulation of COVID-19 and its variants, and novel coronaviruses due to future zoonotic transmissions, combined with the current paucity of coronavirus antivirals, emphasize the need for improved screening in developing effective antivirals for the treatment of infection by SARS-CoV-2 (CoV2) and other coronaviruses. Here we report the development of a live-cell based assay for evaluating the intracellular function of the critical, highly-conserved CoV2 target, the Main 3C-like protease (Mpro). This assay is based on expression of native wild-type mature CoV2 Mpro, the function of which is quantitatively evaluated in living cells through cleavage of a biosensor leading to loss of fluorescence. Evaluation does not require cell harvesting, allowing for multiple measurements from the same cells facilitating quantification of Mpro inhibition, as well as recovery of function upon removal of inhibitory drugs. The pan-coronavirus Mpro inhibitor, GC376, was utilized in this assay and effective inhibition of intracellular CoV2 Mpro was found to be consistent with levels required to inhibit CoV2 infection of human lung cells. We demonstrate that GC376 is an effective inhibitor of intracellular CoV2 Mpro at low micromolar levels, while other predicted Mpro inhibitors, bepridil and alverine, are not. Results indicate this system can provide a highly effective high-throughput coronavirus Mpro screening system.

Gene Annotation in High Schools: Successful Student Pipeline and Teacher Professional Development in Bioscience Using GENI-ACT.

Knowledge of genomics is an essential component of science for high school student health literacy. However, few high school teachers have received genomics training or any guidance on how to teach the subject to their students. This project explored the impact of a genomics and bioinformatics research pipeline for high school teachers and students using an introduction to genome annotation research as the catalyst. The Western New York-based project had three major components: (1) a summer teacher professional development workshop to introduce genome annotation research, (2) teacher-guided student genome annotation group projects during the school year, (3) with an end of the academic year capstone symposium to showcase student work in a poster session. Both teachers and students performed manual gene annotations using an online annotation toolkit known as Genomics Education National Initiative-Annotation Collaboration Toolkit (GENI-ACT), originally developed for use in a college undergraduate teaching environment. During the school year, students were asked to evaluate the data they had collected, formulate a hypothesis about the correctness of the computer pipeline annotation, and present the data to support their conclusions in poster form at the symposium. Evaluation of the project documented increased content knowledge in basic genomics and bioinformatics as well as increased confidence in using tools and the scientific process using GENI-ACT, thus demonstrating that high school students are capable of using the same tools as scientists to conduct a real-world research task.

In silico Analyses of Skin and Peripheral Blood Transcriptional Data in Cutaneous Lupus Reveals CCR2-A Novel Potential Therapeutic Target.

Cutaneous lesions feature prominently in lupus erythematosus (LE). Yet lupus and its cutaneous manifestations exhibit extraordinary clinical heterogeneity, making it imperative to stratify patients with varying organ involvement based on molecular criteria that may be of clinical value. We conducted several in silico bioinformatics-based analyses integrating chronic cutaneous lupus erythematosus (CCLE)-skin and blood expression profiles to provide novel insights into disease mechanisms and potential future therapy. In addition to substantiating well-known prominent apoptosis and interferon related response in both tissue environments, the overrepresentation of GO categories in the datasets, in the context of existing literature, led us to model a "disease road-map" demonstrating a coordinated orchestration of the autoimmune response in CCLE reflected in three phases: (1) initiation, (2) amplification, and (3) target damage in skin. Within this framework, we undertook in silico interactome analyses to identify significantly "over-connected" genes that are potential key functional players in the metabolic reprogramming associated with skin pathology in CCLE. Furthermore, overlapping and distinct transcriptional "hot spots" within CCLE skin and blood expression profiles mapping to specified chromosomal locations offer selected targets for identifying disease-risk genes. Lastly, we used a novel in silico approach to prioritize the receptor protein CCR2, whose expression level in CCLE tissues was validated by qPCR analysis, and suggest it as a drug target for use in future potential CCLE therapy.

Genomic Investigation of Lupus in the Skin.

Lupus erythematosus is a chronic autoimmune disorder with a protean clinical manifestation affecting virtually every organ including skin, with tremendous variation between patients. This makes it vital to stratify patients on a molecular basis. We used gene microarray technology for large-scale screening combined with bioinformatics to investigate global patterns of gene expression in cutaneous lupus erythematosus to allow further insights into disease heterogeneity. Unbiased clustering exposed a clear separation between cutaneous lupus erythematosus skin and blood samples. Pathway-based analyses of the differentially expressed genes from sample groups within skin and blood showed prominent apoptosis and interferon response signals. Given their well-known role in systemic lupus, the two processes are potentially critical to cutaneous lupus erythematosus as well. We found both coincident and distinct features between systemic lupus and cutaneous lupus erythematosus, as well as several pathways and processes that are specific to the cutaneous disease that offer potential therapeutic choices in the future. Finally, we identified shared cutaneous lupus erythematosus-skin and -blood transcriptional "hot spots" located on the genome that include several differentially expressed genes previously associated with the systemic disease. The differentially expressed genes included in the hot spots with no systemic lupus associations can potentially be targeted in future studies aimed at identifying risk genes related to cutaneous disease.

Vitiligo blood transcriptomics provides new insights into disease mechanisms and identifies potential novel therapeutic targets.

BACKGROUND: Significant gaps remain regarding the pathomechanisms underlying the autoimmune response in vitiligo (VL), where the loss of self-tolerance leads to the targeted killing of melanocytes. Specifically, there is incomplete information regarding alterations in the systemic environment that are relevant to the disease state. METHODS: We undertook a genome-wide profiling approach to examine gene expression in the peripheral blood of VL patients and healthy controls in the context of our previously published VL-skin gene expression profile. We used several in silico bioinformatics-based analyses to provide new insights into disease mechanisms and suggest novel targets for future therapy. RESULTS: Unsupervised clustering methods of the VL-blood dataset demonstrate a "disease-state"-specific set of co-expressed genes. Ontology enrichment analysis of 99 differentially expressed genes (DEGs) uncovers a down-regulated immune/inflammatory response, B-Cell antigen receptor (BCR) pathways, apoptosis and catabolic processes in VL-blood. There is evidence for both type I and II interferon (IFN) playing a role in VL pathogenesis. We used interactome analysis to identify several key blood associated transcriptional factors (TFs) from within (STAT1, STAT6 and NF-kB), as well as "hidden" (CREB1, MYC, IRF4, IRF1, and TP53) from the dataset that potentially affect disease pathogenesis. The TFs overlap with our reported lesional-skin transcriptional circuitry, underscoring their potential importance to the disease. We also identify a shared VL-blood and -skin transcriptional "hot spot" that maps to chromosome 6, and includes three VL-blood dysregulated genes (PSMB8, PSMB9 and TAP1) described as potential VL-associated genetic susceptibility loci. Finally, we provide bioinformatics-based support for prioritizing dysregulated genes in VL-blood or skin as potential therapeutic targets. CONCLUSIONS: We examined the VL-blood transcriptome in context with our (previously published) VL-skin transcriptional profile to address a major gap in knowledge regarding the systemic changes underlying skin-specific manifestation of vitiligo. Several transcriptional "hot spots" observed in both environments offer prioritized targets for identifying disease risk genes. Finally, within the transcriptional framework of VL, we identify five novel molecules (STAT1, PRKCD, PTPN6, MYC and FGFR2) that lend themselves to being targeted by drugs for future potential VL-therapy.

Front-end genomics: using an alternative approach for the recovery of high-quality DNA from core needle biopsies.

AIMS: Determine whether a simple prewash step will provide adequate amounts of high-quality DNA from core needle biopsies for molecular sequencing studies. METHODS: The quantitative and qualitative metrics of DNA recovered from core needle biopsies processed either by 1) formalin fixation and paraffin embedding (FFPE), 2) cells recovered after the core needle biopsy was washed, and 3) frozen sections of the core needle biopsy tissue were evaluated and compared to one another. RESULTS: Fairly equivalent amounts of DNA can be obtained from cells recovered from a prewash step relative to the FFPE and frozen section samples. The number of amplifiable DNA in the wash sample was greater than that from the FFPE samples. The average molecular size of DNA in the wash sample was greater than that of both the FFPE and frozen samples. CONCLUSIONS: Although more starting material in terms of the number of cells was present in both the FFPE and frozen section samples than the wash samples, equivalent to better results were obtained from the latter with regard to quality. This approach may be a means to better aliquot the diminutive amounts of tissue associated with core needle biopsies, allowing dissociated cells to be dedicated for molecular studies while keeping the tissue intact for morphological studies.

Increased oxidative stress in pemphigus vulgaris is related to disease activity and HLA-association.

Pemphigus vulgaris (PV) is a rare blistering skin disorder characterized by the disadhesion of keratinocytes due to autoantibody attack against epidermal targets including desmoglein (Dsg) 3, Dsg 1 and possibly other adhesion and non-adhesion molecules. The mechanisms leading to immune-mediated pathology in PV are multifactorial and not fully understood. Recently, oxidative stress (antioxidant/oxidant disequilibrium) has been proposed as a contributory mechanism of autoimmune skin diseases, including PV. In this study, we directly assessed oxidative stress via measurement of total antioxidant capacity (TAC) using ELISA in 47 PV patients, 25 healthy controls and 18 bullous pemphigoid (BP) patients. We also performed microarray gene expression analysis on a separate set of 21 PV patients and 10 healthy controls to evaluate transcriptional dysregulation in oxidative stress-related pathways. Our data indicate that there is a significant reduction in TAC levels in PV patients compared with healthy controls, as well as BP patients. Furthermore, PV patients with active disease have significantly lower TAC levels than PV patients in remission. We also find that HLA allele status has a significant influence on oxidative stress. These findings are corroborated by microarray analysis showing differentially expressed genes involved in oxidative stress between the aforementioned groups. Collectively, our findings provide support for a role of oxidative stress in PV. Whether increased oxidative stress leads to disease manifestation and/or activity, or if disease activity leads to increased oxidative stress remains unknown. Future longitudinal studies may help to further elucidate the relationship between PV and oxidative stress.

Analysis of protein redox modification by hypoxia.

We examined hypoxia-induced changes in global thiol proteome profile in human prostate cancer cells using a BIAM-based display method. We analyzed the kinetics of protein thiol modification by using a pattern recognition algorithm, self-organizing maps (SOM) clustering, and identified the BIAM-labeled proteins by MALDI-TOF and ESI-tandem mass spectrometry. We found 99 out of 215 of total BIAM-labeled proteins were affected by hypoxia treatment and, yet, with diverse patterns and kinetics of redox modification. Our study proved that proteomics analysis employing the BIAM-labeling method can provide valuable information pertaining to global changes in the redox status of proteins in response to hypoxia.