Peter Sehr - Academia.edu (original) (raw)

Papers by Peter Sehr

The retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene ex... more The retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene expression during the cell cycle. Functional inactivation of Rb family proteins leads to loss of cell-cycle control and promotes genome instability and proliferation, which are hallmarks of cancer. Rb proteins share a structural domain, known as the pocket domain, which mediates association with a large number of cellular proteins. A cleft in the pocket domain binds an LxCxE sequence motif in these proteins, many of which function with Rb proteins to co-regulate transcription during quiescence and G1. Proteins from oncogenic DNA viruses also bind this cleft to inactivate Rb family proteins, and the E7 protein from the human papillomavirus has been used as the primary model for understanding LxCxE motif interactions. Evidence presented previously and here demonstrates that the E7 sequence binds tighter and competes with cellular proteins for the LxCxE site, but the molecular basis for the r...

Antiviral Therapy

In this study, we demonstrate that fusion-active virosomes, containing recombinant human papillom... more In this study, we demonstrate that fusion-active virosomes, containing recombinant human papillomavirus type 16 (HPV16) E7 protein antigen, are capable of inducing a robust class I MHC-restricted cytotoxic T-lymphocyte (CTL) response against HPV-transformed tumour cells in a murine model system. Virosomes are reconstituted viral envelopes, which do not contain the genetic material of the native virus. During the reconstitution process, protein antigens can be encapsulated within the virosomes. In the present study, we used virosomes derived from influenza virus. These virosomes retain the cell binding and membrane fusion characteristics of native influenza virus, and have the capacity to deliver encapsulated antigens to the cytosol of antigen-presenting cells through fusion from within acidic endosomes. After immunization of mice with virosomes containing encapsulated HPV16 E7 protein, the animals developed a strong E7–specific CTL response as assessed by 51Cr release measurements a...

Human papillomavirus (HPV) infection is responsible for the development of cervical cancer and it... more Human papillomavirus (HPV) infection is responsible for the development of cervical cancer and its premalignant lesions in women. The virus-encoded oncogene E6 is a promising target for an anti-HPV drug therapy. The authors describe the devel-opment of a homogenous screening assay for inhibitors of the E6 interaction with its cellular target, the E6-associated pro-tein (E6AP), based on AlphaScreen ® technology. The E6 protein was expressed and purified as glutathione S-transferase (GST) fusion protein, and the binding to a biotinylated E6AP peptide was monitored using GST-detecting Acceptor beads coated either with anti-GST antibody or glutathione. After optimization of the assay conditions, a commercial library of 3000 compounds was screened for inhibitors. Active compounds were retested and counterscreened for E6/E6AP specificity using biotinylated GST as a control protein. The results obtained with both types of GST-detecting reagents correlated very well and demonstrated the gre...

Antiviral Therapy, 2006

In this paper we present fusion-active influenza virosomes as a vaccine delivery system capable o... more In this paper we present fusion-active influenza virosomes as a vaccine delivery system capable of efficient induction of CTL, antitumor and humoral responses against encapsulated human papillomavirus type 16 (HPV16) E7 protein. Virosomes are reconstituted viral membranes which do not contain the genetic material of the virus they are derived from. During the reconstitution process protein antigens can be encapsulated in virosomes. Since functionally reconstituted virosomes retain the cell binding and fusion characteristics of the native virus, influenza virosomes deliver their content to the cell cytosol. Here, we studied in vivo induction of immune responses against virosome-encapsulated HPV16 E7 protein in a murine model system. Upon immunization of mice with E7 virosomes the animals developed strong CTL responses. Immunization with virosomes also resulted in E7-specific antibody responses. Tumor challenge experiments demonstrated that immunization with E7 virosomes resulted in p...

Cette invention concerne des nouveaux composes et de nouelles compositions pharmaceutiques ainsi ... more Cette invention concerne des nouveaux composes et de nouelles compositions pharmaceutiques ainsi que leur utilisation pour la fabrication d'un medicament convenant pour le traitement, l'amelioration ou la prevention d'etats pathologiques dus a une infection virale par des virus ARN a simple brin, sens negatif.

Genes & Development, 2021

Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances wi... more Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of oskar mRNA to the posterior pole of the Drosophila oocyte is mediated by Drosophila kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, aTm1. How the proteins cooperate in mRNA transport is unknown. Here, we present the high-resolution crystal structure of a Khc–aTm1 complex. The proteins form a tripartite coiled coil comprising two in-register Khc chains and one aTm1 chain, in antiparallel orientation. We show that aTm1 binds to an evolutionarily conserved cargo binding site on Khc, and mutational analysis confirms the importance of this interaction for mRNA transport in vivo. Furthermore, we demonstrate that Khc binds RNA directly and that it does so via its alternative cargo binding dom...

Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, esp. ... more Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, esp. cancer. First HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement e.g. in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of the other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like spermine or spermidine. Hence, it also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin labelled acetyl spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibi...

G3 Genes|Genomes|Genetics, 2020

N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease.... more N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent...

npj Vaccines, 2020

We performed an independent comparison of neutralizing and cross-neutralizing antibody (ab) level... more We performed an independent comparison of neutralizing and cross-neutralizing antibody (ab) levels seven months after initiation of three-dose, six-month vaccination schedules with the bivalent and quadrivalent human papillomavirus (HPV) vaccines in adolescent Finnish and Indian females, respectively. We used a semi-automated Pseudovirion-Based Neutralization Assay and observed significantly higher HPV16/18 peak ab-levels in bivalent as compared to quadrivalent vaccine recipients. Bivalent vaccine induced cross-neutralizing HPV31/33/45/52/58 antibodies significantly more frequently and to higher levels than the quadrivalent vaccine. The correlation of bivalent vaccine-induced HPV45 ab-levels with HPV16/18 ab-levels was stronger than that of corresponding quadrivalent vaccine-induced ab-levels, suggesting a qualitatively different cross-reactive response. Our findings on the comparison of the immunogenicity of two HPV vaccine tested in two different populations indicate that further ...

FEBS Letters, 2019

Fibroblast Growth Factor 2 (FGF2) is a cell survival factor with crucial functions in tumor-induc... more Fibroblast Growth Factor 2 (FGF2) is a cell survival factor with crucial functions in tumor-induced angiogenesis. Here, we describe a novel time-resolved FGF2 signaling assay based upon live cell imaging of neuroblastoma cells. To validate this system, we tested 8960 small molecules for inhibition of FGF2 signaling with kinetic resolution. Hit compounds were validated in dose-response experiments for FGF2 signaling, FGF receptor antagonism, downstream ERK phosphorylation and FGF2-dependent chemoresistance in a cellular leukemia model system. The new screening system for FGF2 signaling inhibitors has unique features, deselecting compounds with pleiotropic effects on cell proliferation and, along with the experimental pipeline reported, great potential for the discovery of new classes of FGF2 signaling inhibitors that block FGF2 dependent tumor cell survival.

Papillomavirus Research, 2019

Earlier publication from the ongoing multi-centric study of the International Agency for Research... more Earlier publication from the ongoing multi-centric study of the International Agency for Research on Cancer to evaluate less than three doses of the quadrivalent Human Papillomavirus (HPV) vaccine in India amongst unmarried girls demonstrated non-inferior total antibody titres, neutralizing antibody titres and antibody avidity in 2-dose recipients compared to 3-dose recipients at 15-18 years of age (Bhatla et al., 2018) [7]. The number of participants recruited at 15-18 years of age was 1515 and 1795 in the 3-dose and the 2-dose groups respectively. At a median follow-up of 7 years, incident HPV 16/18 infections were detected in 1.6% women receiving two doses and 0.8% women receiving three doses at 15-18 years. Frequency of incident infection was 7.0% in the age-and site-matched unvaccinated women (N = 1484). No persistent infection from HPV 16 was observed in the 2-or 3-dose recipients and one (0.2%) persistent HPV 18 infection was documented,

The discovery of isozyme-selective histone deacetylase (HDAC) inhibitors is critical for understa... more The discovery of isozyme-selective histone deacetylase (HDAC) inhibitors is critical for understanding the biological functions of individual HDACs and for validating HDACs as clinical drug targets. The isozyme HDAC10 contributes to chemotherapy resistance via inhibition of autophagic flux and has recently been described to be a polyamine deacetylase, but no studies directed toward selective HDAC10 inhibitors have been published. Herein, we disclose that the use of two complementary ligand-displacement assays has revealed unexpectedly potent HDAC10 binding of tubastatin A, which has been previously described as a highly selective HDAC6 inhibitor. We synthesized a targeted selection of tubastatin A derivatives and found that a basic amine in the cap group was required for strong HDAC10, but not HDAC6, binding. Only potent HDAC10 binders mimicked HDAC10 knockdown by causing dose-dependent accumulation of acidic vesicles in the BE(2)-C neuroblastoma cell line. Docking of inhibitors int...

Vaccine, Aug 15, 2018

Human papillomavirus (HPV) vaccination is a major strategy for preventing cervical and other ano-... more Human papillomavirus (HPV) vaccination is a major strategy for preventing cervical and other ano-genital cancers. Worldwide HPV vaccination introduction and coverage will be facilitated if a single dose of vaccine is as effective as two or three doses or demonstrates significant protective effect compared to 'no vaccination'. In a multi-centre cluster randomized trial of two vs three doses of quadrivalent HPV vaccination (Gardasil™) in India, suspension of the vaccination due to events unrelated to the study led to per protocol and partial vaccination of unmarried 10-18 year old girls leading to four study groups, two by design and two by default. They were followed up for the primary outcomes of immunogenicity in terms of L1 genotype-specific binding antibody titres, neutralising antibody titres, and antibody avidity for the vaccine-targeted HPV types and HPV infections. Analysis was per actual number of vaccine doses received. This study is registered with ISRCTN, number I...

Papillomavirus research (Amsterdam, Netherlands), Jun 22, 2018

Extending two-dose recommendations of HPV vaccine to girls between 15-18 years will reduce progra... more Extending two-dose recommendations of HPV vaccine to girls between 15-18 years will reduce program cost and improve compliance. Immunogenicity and vaccine targeted HPV infection outcomes were compared between 1795 girls aged 15-18 years receiving two (1-180 days) and 1515 girls of same age receiving three (1-60-180 days) doses. Immunogenicity outcomes in 15-18 year old two-dose recipients were also compared with the 10-14 year old three-dose (N=2833) and two-dose (N=3184) recipients. The 15-18 year old two-dose recipients had non-inferior L1-binding antibody titres at seven months against vaccine-targeted HPV types compared to three-dose recipients at 15-18 years and three-dose recipients at 10-14 years of age. Neutralizing antibody titres at 18 months in 15-18 year old two-dose recipients were non-inferior to same age three-dose recipients for all except HPV 18. The titres were inferior to those in the 10-14 year old three-dose recipients for all targeted types. Frequency of incide...

Nature, 1997

The actin cytoskeleton is regulated by GTP-hydrolysing proteins, the Rho GTPases 1,2 , which act ... more The actin cytoskeleton is regulated by GTP-hydrolysing proteins, the Rho GTPases 1,2 , which act as molecular switches in diverse signal-transduction processes 3. Various bacterial toxins can inactivate Rho GTPases by ADP-ribosylation 1 or glucosylation 4. Previous research has identified Rho proteins as putative targets for Escherichia coli cytotoxic necrotizing factors 1 and 2 (CNF1 and 2) 5,6. These toxins induce actin assembly and multinucleation in culture cells. Here we show that treatment of RhoA with CNF1 inhibits the intrinsic GTPase activity of RhoA and completely blocks GTPase activity stimulated by the Rho-GTPase-activating protein (rhoGAP). Analysis by mass spectrometry and amino-acid sequencing of proteolytic peptides derived from CNF1-treated RhoA indicate that CNF1 induces deamidation of a glutamine residue at position 63 (Gln 63) to give constitutively active Rho protein.

Journal of Cachexia, Sarcopenia and Muscle, 2017

Background Muscle ring finger 1 (MuRF1) is a muscle-specific ubiquitin E3 ligase activated during... more Background Muscle ring finger 1 (MuRF1) is a muscle-specific ubiquitin E3 ligase activated during clinical conditions associated with skeletal muscle wasting. Yet, there remains a paucity of therapeutic interventions that directly inhibit MuRF1 function, particularly in vivo. The current study, therefore, developed a novel compound targeting the central coiled coil domain of MuRF1 to inhibit muscle wasting in cardiac cachexia. Methods We identified small molecules that interfere with the MuRF1-titin interaction from a 130 000 compound screen based on Alpha Technology. A subset of nine prioritized compounds were synthesized and administrated during conditions of muscle wasting, that is, to C2C12 muscle cells treated with dexamethasone and to mice treated with monocrotaline to induce cardiac cachexia. Results The nine selected compounds inhibited MuRF1-titin complexation with IC 50 values <25 μM, of which three were found to also inhibit MuRF1 E3 ligase activity, with one further showing low toxicity on cultured myotubes. This last compound, EMBL chemical core ID#704946, also prevented atrophy in myotubes induced by dexamethasone and attenuated fibre atrophy and contractile dysfunction in mice during cardiac cachexia. Proteomic and western blot analyses showed that stress pathways were attenuated by ID#704946 treatment, including down-regulation of MuRF1 and normalization of proteins associated with apoptosis (BAX) and protein synthesis (elF2B-delta). Furthermore, actin ubiquitinylation and proteasome activity was attenuated. Conclusions We identified a novel compound directed to MuRF1's central myofibrillar protein recognition domain. This compound attenuated in vivo muscle wasting and contractile dysfunction in cardiac cachexia by protecting de novo protein synthesis and by down-regulating apoptosis and ubiquitin-proteasome-dependent proteolysis.

The Journal of biological chemistry, Aug 5, 2016

Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumo... more Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumor-induced angiogenesis. It is secreted by an unconventional secretory mechanism that is based upon direct translocation across the plasma membrane. Key steps of this process are (i) phosphoinositide dependent membrane recruitment, (ii) FGF2 oligomerization and membrane pore formation and (iii) extracellular trapping mediated by membrane proximal heparan sulfate proteoglycans. Efficient secretion of FGF2 is supported by Tec kinase that stimulates membrane pore formation based upon tyrosine phosphorylation of FGF2. Here, we report the biochemical characterization of the direct interaction between FGF2 and Tec kinase as well as the identification of small molecules that inhibit (i) the interaction of FGF2 with Tec, (ii) tyrosine phosphorylation of FGF2 mediated by Tec in vitro and in a cellular context as well as (iii) unconventional secretion of FGF2 from cells. We further demonstrate the ...