Autophagy dysregulation in cell culture and animals models of Spinal Muscular Atrophy

Abnormal autophagy has become a central thread linking neurodegenerative diseases, particularly of the motor neuron. One such disease is spinal muscular atrophy (SMA), a genetic neuromuscular disorder caused by mutations in the SMN1 gene resulting in low levels of Survival Motor Neuron (SMN) protein. Despite knowing the causal protein, the exact intracellular processes that are involved in the selective loss of motor neurons remains unclear. Autophagy induction can be helpful or harmful depending on the situation, and we sought to understand the state of the autophagic response in SMA. We show that cell culture and animal models demonstrate induction of autophagy accompanied by attenuated autophagic flux, resulting in the accumulation of autophagosomes and their associated cargo. Expression of the SMN-binding protein a-COP, a known modulator of autophagic flux, can ameliorate this autophagic traffic jam

Small Molecules in Development for the Treatment of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease resulting from pathologically low levels of survival motor neuron (SMN) protein. The majority of mRNA from the SMN2 allele undergoes alternative splicing and excludes critical codons, causing an SMN protein deficiency. While there is currently no FDA-approved treatment for SMA, early therapeutic efforts have focused on testing repurposed drugs such as phenylbutyrate (2), valproic acid (3), riluzole (6), hydroxyurea (7), and albuterol (9), none of which has demonstrated clinical effectiveness. More recently, clinical trials have focused on novel small-molecule compounds identified from high-throughput screening and medicinal chemistry optimization such as olesoxime (11), CK-2127107, RG7800, LMI070, and RG3039 (17). In this paper, we review both repurposed drugs and small-molecule compounds discovered following medicinal chemistry optimization for the potential treatment of SMA

The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2.

The origin recognition complex (ORC) coordinates a series of events that lead to initiation of DNA strand duplication. As a nuclear double stranded DNA plasmid, the papillomavirus (PV) genome resembles a mini-chromosome in infected cells. To initiate its replication, the viral E2 protein binds to and recruits the E1 DNA helicase at the viral origin. PV genome replication program exhibits three stages: initial amplification from a single genome upon infection to a few copies per cell, a cell cycle linked maintenance phase, and a differentiation dependent late stage where the genome is amplified to thousands of copies. Involvement of ORC or other pre-replication complex (pre-RC) factors has not been described. We report that human PV (HPV) and bovine PV (BPV-1) E2 proteins bind to ORC2, however, ORC2 was not detected at the viral origin. Depletion of ORC2 enhanced PV replication in a transient replication model and in keratinocytes stably maintaining viral episomes, while there was no effect on copy number in a cell line with integrated HPV genomes. Consistent with this, occupancy of E1 and E2 at the viral origin increased following ORC2 silencing. These data imply that ORC2 is not necessary for activation of the PV origin by E1 and E2 but instead suppresses E2 replicative function. Furthermore, we observed that over-expression of HPV E2 decreased ORC2 occupation at two known mammalian origins of replication, suggesting that E2 restricts pre-ORC assembly that could otherwise compete for host replication complexes necessary for viral genome amplification. We infer that the ORC2 complex with E2 restricts viral replication in the maintenance phase of the viral replication program and that elevated levels of E2 that occur during the differentiation dependent amplification stage subvert ORC loading and hence DNA synthesis at cellular origins

Association of human papillomavirus 16 E2 with Rad50-interacting protein 1 enhances viral DNA replication

This work was supported by a Royal Society university research fellowship awarded to J.L.P. (UF110010). K.C.-L. is supported by a Medical Research Council research grant awarded to J.L.P. (MR/N023498/1).Rad50-interacting protein 1 (Rint1) associates with the DNA damage response protein Rad50 during the transition from the S phase to the G2/M phase and functions in radiation-induced G2 checkpoint control. It has also been demonstrated that Rint1 is essential in vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) through an interaction with Zeste-White 10 (ZW10). We have isolated a novel interaction between Rint1 and the human papillomavirus 16 (HPV16) transcription and replication factor E2. E2 binds to Rint1 within its ZW10 interaction domain, and we show that in the absence of E2, Rint1 is localized to the ER and associates with ZW10. E2 expression results in a disruption of the Rint1-ZW10 interaction and an accumulation of nuclear Rint1, coincident with a significant reduction in vesicle movement from the ER to the Golgi apparatus. Interestingly, nuclear Rint1 and members of the Mre11/Rad50/Nbs1 (MRN) complex were found in distinct E2 nuclear foci, which peaked during mid-S phase, indicating that the recruitment of Rint1 to E2 foci within the nucleus may also result in the recruitment of this DNA damage-sensing protein complex. We show that exogenous Rint1 expression enhances E2-dependent virus replication. Conversely, the overexpression of a truncated Rint1 protein that retains the E2 binding domain but not the Rad50 binding domain acts as a dominant negative inhibitor of E2-dependent HPV replication. Put together, these experiments demonstrate that the interaction between Rint1 and E2 has an important function in HPV replication.Publisher PDFPeer reviewe

Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug-like compounds

Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full-length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high-throughput screening assay to identify small molecules that increase the expression of full-length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA

Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53.

Human β-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer

Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

The papillomavirus (PV) E2 protein is a sequence-specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Posttranslational modifications (PTMs) of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine 111 (K111) as a target of p300 acetylation in bovine PV (BPV). The di-lysines at 111 and 112 are conserved in almost all papillomaviruses. We pursued a mutational approach to query the functional significance of lysine in human PV (HPV) E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1-mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. While the replication-defective E2-K111R mutant recruited E1 to the viral replication origin, surprisingly, unwinding of the duplex DNA did not occur. In contrast, the K111 glutamine (K111Q) mutant increased origin melting and stimulated replication compared to wild-type E2. These experiments reveal a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111.IMPORTANCE HPV is one of the most common sexually transmitted infections in the United States. Over 200 HPVs have been described, and they manifest in a variety of ways; they can be asymptomatic or can result in benign lesions (papillomas) or progress to malignancy. Although 90% of infections are asymptomatic and resolve easily, HPV16 and -18 alone are responsible for 70% of all cervical cancers, which are almost entirely caused by HPV infection. Interestingly, 60 to 90% of other cancers have been linked to HPV. The goal of this research is to further elucidate the mechanisms that regulate and mediate viral replication

Characterization of a Conserved Cysteine Residue in the Papillomavirus E2 Protein

Background: Human papillomaviruses (HPVs) are DNA tumor viruses that infect cutaneous and mucosal epithelium. While most infections are self-limiting, a small subset that infects the mucosal epithelium progresses to cancer. All papillomaviruses encode the protein E2 which regulates viral transcription and replication; a highly conserved cysteine residue in the DNA contact helix of E2 plays an unknown role. Previous research suggests the residue is not necessary for replication or binding to DNA. We hypothesize that post-translational modification of this conserved cysteine residue leads to release of viral DNA during packaging of progeny virions. Methods: Mutations of the murine papillomavirus conserved E2 C307 residue to serine and phenylalanine were used to investigate its role in E2 function. C33A, HPV negative cervical cancer cells, were transfected with an E2-responsive luciferase reporter and either wild type or mutant C307 E2 vectors; luciferase assays were performed 48 hours post-transfection to assess transcriptional activity. Whole cell lysates from overexpressed C307 mutants were separated by SDS-PAGE and immunoblotted to assess expression levels relative to wild type. To examine protein localization, C33A cells were transfected with equal amounts of wild type or mutant E2 and fixed 48 hours post-transfection for immunofluorescence. Results: C307S and C307F mutants are both capable of weakly activating transcription. Overexpression of the mutants resulted in a dose dependent increase in transcriptional activity. Both mutants are expressed at levels comparable to wild type E2 and are correctly localized to the nucleus. Conclusion/Impact: The deficient transcription function displayed by the C307 mutants cannot be explained by poor expression or mislocalization. Continued study of this conserved cysteine will help to further understanding of papillomavirus biology and may offer insight into novel avenues for treatment or prevention of HPV-associated cancers

Efficacy of Mutant HPV-16 E6 Proteins in p53 Degradation

Background and Objective:High-risk human papilloma viruses (HPV) cause the majority of anal, cervical, vaginal, vulvar, penile, and oropharyngeal cancers with an annual incidence of 630,000 cases worldwide. HPVs can cause dysplasia that increases neoplasia risk. HPV’s encode eight major viral proteins with the E6 protein being crucial for replication. E6 binding to ubiquitin ligase E6AP initiates polyubiquitination of p53, targeting the protein for proteasomal degradation. We are taking a novel approach to inhibit HPV infection by designing inhibitors targeting the E6-E6AP binding pocket, thereby preventing p53 degradation and restoring its tumor suppressor function. To affirm interaction with the targeted binding site, we generated E6 point mutations that were designed to disrupt interaction with the compound. To ensure their suitability for our studies, we are herein characterizing their capacity to bind E6AP and degrade p53. Methods:E6 mutants were generated by site-directed mutagenesis and confirmed by sequencing. H1299 cells were transfected with GFP, wild-type (WT) E6, or mutant HPV-16 E6 plasmids +/- WT p53 plasmid. After 48 hours, cells were lysed and 16E6 immunoprecipitated. Proteins bound to 16 E6 were separated by SDS-PAGE and subjected to western blot. Binding to E6AP was analyzed and presence of E6 was confirmed by immunoblotting. To test for p53 degradation, H1299 cells were transfected with firefly luciferase (transfection control) and a p53-luciferase fusion gene along with WT and mutant E6, or empty LXSN plasmid. 48 hours later p53- luciferase was measured with Dual-Glo Luciferase Assay. Results:Western blot of immunoprecipitation lysates revealed that the 16E6(Y32F) mutant retained E6AP binding capacity. p53 degradation assay showed p53 degradation comparable to WT for both 16E6(Y32F) and 16E6(C51S) mutant proteins. Conclusion:Our findings indicate the 16E6(Y32F) mutant will be acceptable for use in future compound studies, and p53 degradation ability of 16E6(C51S) implies that this also retains E6AP binding

Effect of Tyrosine Phosphorylation of HPV31 E2 on Replication

Background  The HPV early E2 protein is an essential regulatory protein involved in HPV replication and transcription that is encoded by all papillomaviruses. Phosphorylation of E2 at Y102 inhibits viral replication, and an additional phosphorylation site at Y87 was recently identified through mass spectrometry. This project aimed to characterize E2 proteins encoding mutations at Y87 that abrogate phosphorylation. In parallel, we aimed to identify the kinase responsible for E2 phosphorylation at Y87. The Androphy lab previously has published that FGFR tyrosine kinases effect HPV replication, but there may be other kinases which act upon E2. Tyk2 is a tyrosine kinase regulated by the viral protein E6 and highly expressed in keratinocytes. We want to determine if Tyk2 is involved in regulation of E2 through phosphorylation.  Experimental Design  We will co-express Tyk2 and E2 in 293TT cells and through immunoprecipitations determine if Tyk2 binds E2. Using an in-vitro replication assay, viral proteins E2 and E1 will be overexpressed along with Tyk2 to determine if Tyk2 effects replication. We will make HPV genomes with Y87E/Y87F mutations through site-directed mutagenesis. Expression of mutant E2 will be examined through transfection of cells and western blot analysis.  Results  E2 harboring 87 to glutamic acid (Y87E) or phenylalanine (Y87F) mutations produces soluble protein, but 87E inhibits replication. Tyk2 expression stabilizes E2 suggesting it binds E2.  Potential impact  It is important to understanding the mechanisms underlying a virus’ replication cycle for future targeted treatments against infection