Lehrstuhl für Biochemie
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- CIBSS Centre for Integrative Biological Signalling Studies, University of Freiburg (1)
- Genelux Corporation, San Diego Science Center, 3030 Bunker Hill Street, Suite 310, San Diego, California 92109, USA (1)
- MRB Forschungszentrum für Magnet-Resonanz-Bayern e.V., Am Hubland, D-97074 Würzburg (1)
- Rudolf-Virchow-Zentrum DFG-Forschungszentrum für Experimentelle Biomedizin der Universität Würzburg (1)
Background
ACAM2000, a thymidine kinase (TK)-positive strain of vaccinia virus, is the current smallpox vaccine in the US. Preclinical testing demonstrated potent oncolytic activity of ACAM2000 against several tumor types. This Phase I clinical trial of ACAM2000 delivered by autologous adipose stromal vascular fraction (SVF) cells was conducted to determine the safety and feasibility of such a treatment in patients with advanced solid tumors or acute myeloid leukemia (AML).
Methods
Twenty-four patients with solid tumors and two patients with AML participated in this open-label, non-randomized dose-escalation trial. All patients were treated with SVF derived from autologous fat and incubated for 15 min to 1 h with ACAM2000 before application. Six patients received systemic intravenous application only, one patient received intra-tumoral application only, 15 patients received combination intravenous with intra-tumoral deployment, 3 patients received intravenous and intra-peritoneal injection and 1 patient received intravenous, intra-tumoral and intra-peritoneal injections. Safety at each dose level of ACAM2000 (1.4 × 106 plaque-forming units (PFU) to 1.8 × 107 PFU) was evaluated. Blood samples for PK assessments, flow cytometry and cytokine analysis were collected at baseline and 1 min, 1 h, 1 day, 1 week, 1 month, 3 months and 6 months following treatment.
Results
No serious toxicities (> grade 2) were reported. Seven patients reported an adverse event (AE) in this study: self-limiting skin rashes, lasting 7 to 18 days—an expected adverse reaction to ACAM2000. No AEs leading to study discontinuation were reported. Viral DNA was detected in all patients’ blood samples immediately following treatment. Interestingly, in 8 patients viral DNA disappeared 1 day and re-appeared 1 week post treatment, suggesting active viral replication at tumor sites, and correlating with longer survival of these patients. No major increase in cytokine levels or correlation between cytokine levels and skin rashes was noted. We were able to assess some initial efficacy signals, especially when the ACAM2000/SVF treatment was combined with checkpoint inhibition.
Conclusions
Treatment with ACAM2000/SVF in patients with advanced solid tumors or AML is safe and well tolerated, and several patients had signals of an anticancer effect. These promising initial clinical results merit further investigation of therapeutic utility.
Trial registration Retrospectively registered (ISRCTN#10201650) on October 22, 2018.
Background
Previous studies have identified IFNγ as an important early barrier to oncolytic viruses including vaccinia. The existing innate and adaptive immune barriers restricting oncolytic virotherapy, however, can be overcome using autologous or allogeneic mesenchymal stem cells as carrier cells with unique immunosuppressive properties.
Methods
To test the ability of mesenchymal stem cells to overcome innate and adaptive immune barriers and to successfully deliver oncolytic vaccinia virus to tumor cells, we performed flow cytometry and virus plaque assay analysis of ex vivo co-cultures of stem cells infected with vaccinia virus in the presence of peripheral blood mononuclear cells from healthy donors. Comparative analysis was performed to establish statistically significant correlations and to evaluate the effect of stem cells on the activity of key immune cell populations.
Results
Here, we demonstrate that adipose-derived stem cells (ADSCs) have the potential to eradicate resistant tumor cells through a combination of potent virus amplification and sensitization of the tumor cells to virus infection. Moreover, the ADSCs demonstrate ability to function as a virus-amplifying Trojan horse in the presence of both autologous and allogeneic human PBMCs, which can be linked to the intrinsic immunosuppressive properties of stem cells and their unique potential to overcome innate and adaptive immune barriers. The clinical application of ready-to-use ex vivo expanded allogeneic stem cell lines, however, appears significantly restricted by patient-specific allogeneic differences associated with the induction of potent anti-stem cell cytotoxic and IFNγ responses. These allogeneic responses originate from both innate (NK)- and adaptive (T)- immune cells and might compromise therapeutic efficacy through direct elimination of the stem cells or the induction of an anti-viral state, which can block the potential of the Trojan horse to amplify and deliver vaccinia virus to the tumor.
Conclusions
Overall, our findings and data indicate the feasibility to establish simple and informative assays that capture critically important patient-specific differences in the immune responses to the virus and stem cells, which allows for proper patient-stem cell matching and enables the effective use of off-the-shelf allogeneic cell-based delivery platforms, thus providing a more practical and commercially viable alternative to the autologous stem cell approach.
Interleukin 2 (IL-2) was the first cytokine applied for cancer treatment in human history. It has been approved as monotherapy for renal cell carcinoma and melanoma by the FDA and does mediate the regression of the tumors in patients. One of the possible mechanisms is that the administration of IL-2 led to T lymphocytes expansion, including CD4+ and CD8+ T cells. In addition, a recent study demonstrated that antigen-specific T cells could also be expanded through the induction of IL-2, which plays a crucial role in mediating tumor regression. However, despite the long-term and extensive use of IL-2 in the clinic, the ratio of patients who get a complete response was still low, and only about one-fifth of patients showed objective tumor regression. Therefore, the function of IL-2 in cancer treatment should continue to be optimized and investigated. A study by Franz O. Smith et al. has shown that the combination treatment of IL-2 and tumor-associated antigen vaccine has a strong trend to increased objective responses compared to patients with melanoma receiving IL-2 alone. Peptide vaccines are anti-cancer vaccines able to induce a powerful tumor antigenspecific immune response capable of eradicating the tumors. According to the type of antigens, peptide vaccines can be classified into two distinct categories: Tumor-associated antigens (TAA) vaccine and tumor-specific neoantigens (TSA) vaccine. Currently, Peptide vaccines are mainly investigated in phase I and phase II clinical trials of human cancer patients with various advanced cancers such as lung cancer, gastrointestinal tumors, and breast cancers. Vaccinia virus (VACV) is one of the safest viral vectors, which has been wildly used in cancer treatment and pathogen prevention. As an oncolytic vector, VACV can carry multiple large foreign genes, which enable the virus to introduce diagnostic and therapeutic agents without dramatically reducing the viral replication. Meanwhile, the recombinant vaccinia virus (rVACV) can be easily generated by homologous recombination. Here, we used the vaccinia virus as the therapeutic cancer vector, expressing mouse Interleukin 2 (IL-2) and tumor-associated antigens simultaneously to investigate the combined effect of anti-tumor immune response in the 4T1 mouse tumor model. As expected, the VACV driven mIL-2 expression remarkably increased both CD4+ and CD8+ populations in vivo, and the virus-expressed tumor-associated peptides successfully elicited theantigen-specific T cell response to inhibit the growth of tumors. Furthermore, the experiments with tumor-bearing animals showed that the mIL-2 plus tumor antigens expressing VACV vector gave a better anti-cancer response than the mIL-2 alone expressing vector. The combinations did significantly more inhibit tumor growth than mIL-2 treatment alone. Moreover, the results confirmed our previous unpublished data that the mIL-2 expression driven by synthetic early/late promoter in the Lister strain VACV could enhance the tumor regression in the 4T1 mouse model.
Im Zellkern eukaryotischer Zellen werden Gene in mRNAs transkribiert, welche umfangreich prozessiert und aus dem Zellkern exportiert werden. Im Zytoplasma erfolgt die Translation der mRNAs in Proteine, ein Prozess, welcher viel Energie benötigt und daher mittels vielfältiger Mechanismen streng reguliert wird. Ein Beispiel hierfür stellt die Klasse der TOP-mRNAs dar, eine RNA-Spezies, welche hauptsächlich Transkripte von Genen umfasst, die selbst in die Translation involviert sind. Die prominentesten Vertreter dieser Klasse sind die Proteine der kleinen und großen ribosomalen Untereinheiten. TOP-mRNAs zeichnen sich durch ein gemeinsames Sequenz-Motiv am Anfang Ihrer 5’-UTR aus, welches aus einem Pyrimidinstrang besteht und unmittelbar nach dem Cap mit einem Cytosin beginnt. Dieses allen TOP-RNAs gemeinsame Motiv ermöglicht die zeitgleiche Translationskontrolle dieser RNA-Klasse. So kann die Translation der TOP-mRNAs unter Stressbedingungen wie z.B. Nährstoffmangel koordiniert inhibiert werden, wodurch Energie eingespart wird.
Bereits lange wird nach einem Regulator gesucht, der an dieses TOP-Motiv bindet und die koordinierte Regulation ermöglicht. Man kann sich hier einen Inhibitor oder auch einen Aktivator vorstellen. Verschiedene Proteine wurden bereits in Erwägung gezogen. In dieser Arbeit wurde das Protein TIAR mittels Massenspektrometrie als TOP-interagierender Faktor identifiziert und dessen Bindungseigenschaften mit dem TOP-Motiv durch Shift Assays untersucht. Hierbei konnten Minimalkonstrukte verschiedener Organismen sowie RNA-TOP – Sequenzen identifiziert werden, welche sich für Strukturanalysen eignen würden. Als weiterer TOP-interagierender Faktor wurde über verschiedene sequenzielle Reinigungsschritte das Protein 14-3-3ε identifiziert.
Weiterhin wurden die TOP-Motiv-bindenden Proteine LARP1 und LARP7 auf Ihre Bindungseigenschaften mit Ihren Zielsequenzen untersucht. Während gezeigt werden konnte, dass LARP1 einen inhibierenden Einfluss auf TOP-RNAs hat, wurde in weiteren Shift-Assays die Bindungseigenschaften von LARP7 mit 7SK untersucht, wobei ebenfalls ein minimales LARP7–Konstrukt sowie 7SK-Konstrukte für Strukturanalysen identifiziert werden konnten. Weiterhin konnte gezeigt werden, dass verschiedene Substanzen wie tRNA und Arginin einen starken Einfluss auf die LARP7-7SK – Interaktion ausüben, welcher in weiteren Studien berücksichtigt werden sollte.
Das kolorektale Karzinom ist eines der häufigsten beim Menschen vorkommenden
Karzinome [2]. Diesem liegen unterschiedliche Mutationen zugrunde, die in knapp 100%
der kolorektalen Karzinome zu einer Überexpression von MYC führen, welches als
Transkriptionsfaktor maßgeblich den Zellzyklus, Proliferation und Vaskularisierung
beeinflusst [10,16]. Damit stellt MYC ein potenzielles Therapieziel in der Behandlung des
Kolorektalen Karzinoms dar. Zusätzlich konnte in den letzten Jahren ein Onkoprotein
namens CIP2A identifiziert werden, welches nach Depletion mit einem Verlust von MYC
Protein einhergeht [69]. Zusätzlich ist CIP2A ein unabhängiger prognostischer Faktor im
Kolorektalen Karzinom [70].
Diese Arbeit konnte zeigen, dass CIP2A-depletierte Zellen einen deutlichen
Wachstumsnachteil gegenüber unbehandelten Zellen zeigen. Dieser Unterschied kann nicht
durch eine gesteigerte Apoptose, sondern vielmehr durch einen verlängerten Zellzyklus
erklärt werden. Weiterhin konnte eine neue Zelllinie mit DOX-induzierbarer shCIP2A
hergestellt werden, die für weitere Experimente genutzt werden kann. Entgegen der
Wirkweise im Zervixkarzinom [69], konnte im kolorektalen Karzinom kein Einfluss auf die
Stabilität von MYC Protein durch CIP2A nachgewiesen werden. Auch konnte der Verlust
von MYC nach CIP2A Knockdown nicht durch gleichzeitige Inhibierung des Abbaus, durch
Okadasäure, MG132 oder in den FBWX7-defizienten Zellen, verhindert werden. Stattdessen
resultiert die Herunterregulation von CIP2A in einem leichten Rückgang der MYC-mRNA
Menge und einem deutlichen Verlust an MYC-Protein. In Zellen mit verschiedenen
Konstrukten der MYC Transkripte kann dieser Verlust an MYC Protein auf eine
translationelle Regulation in der 5’UTR zurückgeführt werden, was eine bisher nicht
beschriebene Wirkweise von CIP2A darstellt. Da CIP2A in normalen Zellen praktisch nicht
exprimiert ist [78], könnte dies ein mögliches Ziel in der Tumortherapie darstellen. Dieses
gilt es in weiteren Experimenten noch genauer zu untersuchen.
Die Krebstherapie und Behandlung von Tumoren stellt für die moderne Medizin auch in Zukunft eine enorme Herausforderung dar. Trotz intensiver Forschung konnten in den letzten Jahrzehnten zwar zunehmend Fortschritte erzielt werden, allerdings muss das Spektrum an neuen Therapieformen und Möglichkeiten kontinuierlich erweitert werden. In den letzten Jahren haben die Kalorienrestriktion sowie die Aminosäuren- und Proteinrestriktion zunehmend an Bedeutung gewonnen, da sie einen erheblichen positiven Einfluss auf die Entstehung von altersassoziierten Erkrankungen wie z.B. Krebs haben. Allen Formen gemeinsam ist die Induktion eines Low-Energy-Metabolismus, der die Zellen in einen antiproliferativen und selbst-regenerierenden Zustand versetzt. In dieser Arbeit sollte untersucht werden, ob die Methionin-Restriktion als eine Form der Aminosäurerestriktion sich grundsätzlich als Therapieform im Plattenepithelkarzinom (HNSCC) eignet. Zusätzlich sollte ein einfaches zelluläres Modellsystem etabliert werden, das auf metaboler Ebene die Charakterisierung und Analyse des Low-Energy-Metabolismus ermöglicht. Es konnte aufgezeigt werden, dass die Methionin-Restriktion eine effektive Methode ist, um die Proliferation ausgesuchter Zelllinien des HNSCC zu inhibieren. Des Weiteren konnte aufgezeigt werden, dass der Einsatz von Aminosäure-Analoga eine weitere Möglichkeit darstellt, auf die Proliferation von Tumorzellen Einfluss zu nehmen. Die massenspektrometrische Analyse der murinen Zelllinie L929 mittels LC/MS lieferte über einen Zeitraum von 5 Tagen ein detailliertes Bild des Stoffwechsels von mehr als 150 Metaboliten unter Methionin-Restriktion. Durch die Definition eines charakteristischen Fingerabdrucks nach 48 h und eines nur wenige Metabolite umfassenden Fußabdrucks konnte ein murines Modellsystem etabliert werden, dass die Analyse von potentiellen Wirkstoffen, u.a. sogenannten caloric restriction mimetics, ermöglicht.
∆Np63 is a master regulator of squamous cell identity and regulates several signaling pathways that crucially
contribute to the development of squamous cell carcinoma (SCC) tumors. Its contribution to coordinating the
expression of genes involved in oncogenesis, epithelial identity, DNA repair, and genome stability has been
extensively studied and characterized. For SCC, the expression of ∆Np63 is an essential requirement to
maintain the malignant phenotype. Additionally, ∆Np63 functionally contributes to the development of cancer
resistance toward therapies inducing DNA damage.
SCC patients are currently treated with the same conventional Cisplatin therapy as they would have been
treated 30 years ago. In contrast to patients with other tumor entities, the survival of SCC patients is limited,
and the efficacy of the current therapies is rather low. Considering the rising incidences of these tumor entities,
the development of novel SCC therapies is urgently required. Targeting ∆Np63, the transcription factor, is a
potential alternative to improve the therapeutic response and clinical outcomes of SCC patients.
However, ∆Np63 is considered “undruggable.” As is commonly observed in transcription factors, ∆Np63 does
not provide any suitable domains for the binding of small molecule inhibitors. ∆Np63 regulates a plethora of
different pathways and cellular processes, making it difficult to counteract its function by targeting
downstream effectors. As ∆Np63 is strongly regulated by the ubiquitin–proteasome system (UPS), the
development of deubiquitinating enzyme inhibitors has emerged as a promising therapeutic strategy to target
∆Np63 in SCC treatment.
This work involved identifying the first deubiquitinating enzyme that regulates ∆Np63 protein stability. Stateof-the-art SCC models were used to prove that USP28 deubiquitinates ∆Np63, regulates its protein stability,
and affects squamous transcriptional profiles in vivo and ex vivo. Accordingly, SCC depends on USP28 to
maintain essential levels of ∆Np63 protein abundance in tumor formation and maintenance. For the first time,
∆Np63, the transcription factor, was targeted in vivo using a small molecule inhibitor targeting the activity of
USP28. The pharmacological inhibition of USP28 was sufficient to hinder the growth of SCC tumors in
preclinical mouse models.
Finally, this work demonstrated that the combination of Cisplatin with USP28 inhibitors as a novel therapeutic
alternative could expand the limited available portfolio of SCC therapeutics. Collectively, the data presented
within this dissertation demonstrates that the inhibition of USP28 in SCC decreases ∆Np63 protein abundance,
thus downregulating the Fanconi anemia (FA) pathway and recombinational DNA repair. Accordingly, USP28
inhibition reduces the DNA damage response, thereby sensitizing SCC tumors to DNA damage therapies, such
as Cisplatin.
Cancer is one of the major causes of mortality in developed countries. In 2020, there were more than 19.3 million new cases of tumor malignancies worldwide, with more than 10 million deaths. The high rates of cancer cases and mortality necessitate extensive research and the development of novel cancer treatments and antitumor agents. In most cases, conventional treatment strategies for tumor therapy are based on chemotherapeutic treatment, which is supplemented with radiotherapy and/or surgical resection of solid tumors [1]. The use of chemotherapy for the treatment of cancer has significant side effects, the most dangerous of which is toxicity [2] [3].
Modern methods of treating tumors focus on specific drug delivery to the tumor site, actively targeting the tumor cells, as well as the reduction of side effects. One of the most promising current approaches is based on oncolytic viruses. Antitumor properties of viruses were documented at the beginning of the 20th century when some cancer patients recovered after acute viral infections, particularly influenza [4]. Vaccinia virus (VACV) is a member of the Poxviridae family, has natural antitumor properties, and provides a good basis for generating efficient recombinant oncolytic strains. Furthermore, VACV has never been shown to integrate into the host genome [5]. VACV is likely one of the safest and well-studied viruses due to extensive research being done in molecular biology and pathophysiology to investigate its potential as a vaccine for smallpox eradication programs. It has been administered to over 200 million people worldwide. VACV antitumor therapeutic effectiveness has been established in xenograft models with a variety of tumor types for human and canine cancers. Furthermore, recombinant oncolytic VACVs expressing genes encoding light-emitting proteins are a big improvement in a treatment strategy that combines tumor-specific therapies and diagnostics.
Oncolytic virus treatments are effective in xenograft cancer models in mice, however, the significant improvements found in mice do not always translate to human cancer patients. These therapies should be tested in dogs with spontaneous cancer not only to offer well translatable information regarding the possible efficiency of viral therapy for human cancers but also to improve the health of our household pets as well. Spontaneous canine tumors are starting to be regarded as an essential model of human cancers that can reproduce the tumor microenvironment and immune response of cancer patients [6]. Just as data obtained in dog experiments can improve cancer therapy for human patients, these findings can also be used to improve treatment protocols in canine patients.
Hundreds of studies and dozens of reviews have been published regarding the antitumor effects of various recombinants of VACV, but information on the anticancer features of initial, genetically-unmodified “naïve” VACV is still limited. In the first studies, we compared different wild-type, non-modified strains of VACV and tested their oncolytic properties on a panel of various cancer cells derived from different organs. In addition, we also tested a protection system based on the “Trojan horse” concept - using a combination of human Adipose tissue-derived Stem Cells (hADSC) and three different wild-type single plaque purified Vaccinia virus strains: W1, L1, and T1. We showed that all tested human cell lines (FaDu, MDA MB 231, HNT-13, HNT-35, and PC-3) are permissive to L0, W0, T0, L1, W1, and L1 infection. Furthermore, we tested the cytotoxicity of VACV in different cancer cell lines (A549, PC-3, MDA-MB 231, FaDu, HNT-13, HNT-25, and HNT-35). All strains lysed the cells, which was most visible at 96 hpi. We also showed that all tested strains could efficiently infect and multiply in hADSC at a high level. In our in vivo study, we tested the therapeutic efficacy of the wild-type Vaccinia viruses L1, W1, and T1 alone or in combination with hADSC. Wild-type VACV strains were tested for their oncolytic efficiency in human lung adenocarcinoma (A549) in a xenograft model. Treatment of A549 tumors with different doses of L1 and W1 as well as with a L1/ADSC or W1/ADSC combination led to significant tumor regression compared to the PBS control. Additionally, the treatment with L1 and W1 and the combination of L1/ADSC and W1/ADSC was well tolerated by the animals. In the case of the wild-type Tian Tan strain, results were not obtained due to the high cytotoxicity of this strain. Therefore, it should be attenuated for further studies.
In the second part of the current study, we investigated the oncolytic effect of C1-opt1, W1 opt1, and L3-opt1 strains based on the wild-type Copenhagen, Wyeth, and Lister vaccines with additional expression of turboFP635. Replication and cytotoxicity assays demonstrated that all 3 viruses were able to infect, replicate in and kill canine tumor cell lines STSA-1 and CT1258 in a virus dose- and time- dependent fashion. Cytotoxicity and replication assays were also performed on cultured canine Adipose-derived Mesenchymal Stem Cells (cAdMSC). The results showed that the cells were lysed much slower than the tumor cells. It suggests that these cells can harbour the virus for a long-term period, allowing the virus to spread into the body and there is enough time to reach the primary tumor or metastases before the cell carrier is destroyed. The viral replication in cAdMSC in our study was lower than in canine cancer cells (STSA-1 and CT1258) at the same MOI. After being studied in cell culture, C1 opt1 and their combination with cAdMSC (C1-opt1/cAdMSC) were used in canine STSA 1 tumor bearing nude mice. We tested the oncolytic effect of the C1-opt1 virus alone and in combination with cAdMSC in the canine STSA-1 xenograft mouse model. Altogether, our findings have shown that both C1-opt1 and cAdMSC/C1-opt1 significantly reduced tumor size or eliminated the tumor. There was no significant difference between C1-opt1 alone and cAdMSC/C1-opt1. The virus particles were mostly found within the tumor after 24 dpi, some amount of virus particles were found in the lungs of mice injected with a combination of cAdMSC/C1-opt1 but not in the group injected with virus alone (cAdMSC might get stuck in the lungs and cause virus propagation there).
Taken together, this study provided a proof-of-concept that hADSC/cAdMSC can be used as a carrier system for the “Trojan horse” concept. However, it should be confirmed in another experimental model system, such as canine patients. Moreover, these findings suggest that wild-type, non-modified strains of Vaccinia virus isolates can be considered promising candidates for oncolytic virotherapy, especially in combination with mesenchymal stem cells.
The macromolecular SMN complex facilitates the formation of Sm-class ribonucleoproteins involved in mRNA processing (UsnRNPs). While biochemical studies have revealed key activities of the SMN complex, its structural investigation is lagging behind. Here we report on the identification and structural determination of the SMN complex from the lower eukaryote Schizosaccharomyces pombe, consisting of SMN, Gemin2, 6, 7, 8 and Sm proteins. The core of the SMN complex is formed by several copies of SMN tethered through its C-terminal alpha-helices arranged with alternating polarity. This creates a central platform onto which Gemin8 binds and recruits Gemins 6 and 7. The N-terminal parts of the SMN molecules extrude via flexible linkers from the core and enable binding of Gemin2 and Sm proteins. Our data identify the SMN complex as a multivalent hub where Sm proteins are collected in its periphery to allow their joining with UsnRNA.
The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell.
IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.
Bacteria thrive and survive in many different environments, and as a result, they have developed robust mechanisms to adapt rapidly to alterations in their surroundings. The protection against osmotic forces is provided by mechanosensitive channels: their primary function is to maintain the integrity of the cell upon a hypoosmotic shock. The mechanosensitive channel of small conductance (MscS) is not only the smallest common structural unit of a diverse family that allows for a tailored response in osmoregulation; it is also the most intensively studied homologue. Mechanosensitive channels directly sense elevated membrane tension levels generated by increased pressure within the cell and open transiently. Escherichia coli has six paralogues that differ in their gating properties and the number of additional transmembrane (TM) helices. These TM helices, termed sensor paddles, are essential for sensing, as they directly contact the surrounding membrane; however, the role of the additional TM helices is still unclear. Furthermore, lipids occupy hydrophobic pockets far away from the membrane plane. A recent gating model for MscS states that increased membrane tension triggers the expulsion of lipids out of those pockets, modulating different conformational states of MscS. This model focuses on bound lipids, but it is still unclear to what extent the direct interaction with the membrane influences sensing and how relevant it is for the larger paralogues.
In the herein described work, structural studies on two larger paralogues, the medium-sized channel YnaI and the large channel YbiO were realised using electron cryomicroscopy (cryo-EM). Lipids were identified in YnaI in the pockets in a similar position and orientation as in MscS, suggesting a conserved sensing mechanism. Moreover, the copolymer diisobutylene/maleic acid (DIBMA) allowed the extraction of artificially activated YnaI from plasma membranes, leading to an open-like form of this channel. This novel conformation indicated that the pore helices bend at a GGxGG motif during gating, which is unique among the Escherichia coli paralogues, concomitant with a structural reorganisation of the sensor paddles. Thus, despite a high similarity of their closed states, the gating mechanisms of MscS and YnaI are surprisingly different. Furthermore, the comparison of MscS, YnaI, and YbiO accentuates variations and similarities between the differently sized family members, implying fine-tuning of channel properties in the pore regions and the cytosolic lateral entry sides into the channel. Structural analyses of MscS reconstituted into different systems showed the advantages and disadvantages of certain polymers and detergents. The novel DIBMA copolymer and the more conventional amphiphilic polymers, so-called Amphipols, perturb contacting transmembrane helices or lead to their denaturation. Due to this observation, the obtained structures of YnaI must also be cautiously considered. The structures obtained in detergents resulted in unaffected channels; however, the applicability of detergents for MscS-like channels is limited by the increased required sample concentration.
The role of lipids for gating MscS in the absence of a membrane was examined by deliberately removing coordinated lipid molecules from MscS using different amounts and kinds of detergent. The effects on the channel were inspected by cryo-EM. These experiments showed that closed MscS adopts the open conformation when it is enough delipidated by incubation with the detergent n-dodecyl-β-D-maltoside, and adding lipids to the open channel reverses this process. The results agree with the state-of-the-art model that the amount of lipid molecules in the pockets and grooves is responsible for the conformational state of MscS. Furthermore, incubation with the detergent lauryl maltose neopentyl glycol, which has stabilising and delipidating characteristics, resulted in a high-resolution structure of open MscS exhibiting an intricate network of ligands. Based on this structure, an updated gating model is proposed, which states that upon opening, lipids from the pockets migrate into the cytosolic membrane leaflet, while lipids from the periplasmic leaflet enter the grooves that arise between the sensor paddles.
Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a novel cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defense and drive the latent-lytic switch. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective pri-miRNA hairpin loops. Subsequent loss of miR-30 and activation of miR-30/p53/Drp1 axis triggers a profound disruption of mitochondrial architecture, which impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 was sufficient to trigger virus reactivation from latency thereby identifying it as a readily drugable master regulator of the herpesvirus latent-lytic switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 provides exciting therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders like myalgic encephalitis/chronic fatigue syndrome (ME/CFS) and Long-COVID.
Stress granules (SGs) are cytoplasmic condensates containing untranslated mRNP complexes. They are induced by various proteotoxic conditions such as heat, oxidative, and osmotic stress. SGs are believed to protect mRNPs from degradation and to enable cells to rapidly resume translation when stress conditions subside. SG dynamics are controlled by various posttranslationalmodifications, but the role of the ubiquitin system has remained controversial. Here, we present a comparative analysis addressing the involvement of the ubiquitin system in SG clearance. Using high-resolution immuno-fluorescence microscopy, we found that ubiquitin associated to varying extent with SGs induced by heat, arsenite, H2O2, sorbitol, or combined puromycin and Hsp70 inhibitor treatment. SG-associated ubiquitin species included K48- and K63-linked conjugates, whereas free ubiquitin was not significantly enriched. Inhibition of the ubiquitin activating enzyme, deubiquitylating enzymes, the 26S proteasome and p97/VCP impaired the clearance of arsenite- and heat-induced SGs, whereas SGs induced by other stress conditions were little affected. Our data underline the differential involvement of the ubiquitin system in SG clearance, a process important to prevent the formation of disease-linked aberrant SGs.
Gene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here, we report an association of the Smn complex, which mediates spliceosomal snRNP biogenesis, with the 7SK complex involved in transcriptional regulation. We found that Smn interacts with the 7SK core components Larp7 and Mepce and specifically associates with 7SK subcomplexes containing hnRNP R. The association between Smn and 7SK complexes is enhanced upon transcriptional inhibition leading to reduced production of snRNPs. Taken together, our findings reveal a functional association of Smn and 7SK complexes that is governed by global changes in transcription. Thus, in addition to its canonical nuclear role in transcriptional regulation, 7SK has cytosolic functions in fine-tuning spliceosome production according to transcriptional demand.
Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains.
The biogenesis of spliceosomal UsnRNPs is a highly elaborate cellular process that occurs both in the nucleus and the cytoplasm. A major part of the process is the assembly of the Sm-core particle, which consists of a ring shaped heptameric unit of seven Sm proteins (SmD1•D2•F•E•G•D3•B) wrapped around a single stranded RNA motif (termed Sm-site) of spliceosomal UsnRNAs. This process occurs mainly in the cytoplasm by the sequential action of two biogenesis factors united in PRMT5- and SMN-complexes, respectively. The PRMT5-complex composed of the three proteins PRMT5, WD45 and pICln is responsible for the symmetric dimethylation of designated arginine residues in the C-terminal tails of some Sm proteins. The action of the PRMT5- complex results in the formation of assembly incompetent Sm-protein intermediates sequestered by the assembly chaperone pICln (SmD1•D2•F•E•G•pICln and pICln•D3•B). Due to the action of pICln, the Sm proteins in these complexes fail to interact with UsnRNAs to form the mature Sm-core. This kinetic trap is relieved by the action of the SMN-complex, which removes the pICln subunit and facilitates the binding of the Sm-core intermediates to the UsnRNA, thus forming the mature Sm-core particle. The human SMN complex consists of 9 subunits termed SMN, Gemin2-8 and Unrip. So far, there are no available atomic structures of the whole SMN-complex, but structures of isolated domains and subunits of the complex have been reported by several laboratories in the past years. The lack of structural information about the entire SMN complex most likely lies in the biophysical properties of the SMN complex, which possesses an oligomeric SMN core, and many unstructured and flexible regions. These were the biggest roadblocks for its structural elucidation using traditional methods such as X-ray crystallography, NMR or CryoEM. To circumvent these obstacles and to obtain structural insight into the SMN-complex, the Schizosaccharomyces pombe SMN complex was used as a model system in this work. In a collaboration with the laboratory of Dr. Remy Bordonne (IGMM, CNRS, France), we could show that the SpSMN complex is minimalistic in its composition, consisting only of SpSMN, SpGemin2, SpGemin8, SpGemin7 and SpGemin6. Using biochemical experiments, an interaction map of the SpSMN complex was established which was found to be highly similar to the reported map of the human SMN complex. The results of this study clearly show that SpSMN is the oligomeric core of the complex and provides the binding sites for the rest of the subunits. Through biochemical and X-ray scattering experiments, the properties of the SpSMN subunit such as oligomerization viii and intrinsic disorder, were shown to determine the overall biophysical characteristics of the whole complex. The structural basis of SpSMN oligomerization is presented in atomic detail which establishes a dimeric SpSMN as the fundamental unit of higher order SpSMN oligomers. In addition to oligomerization, the YG-box domain of SpSMN serves as the binding site for SpGemin8. The unstructured region of SpSMN imparts an unusual large hydrodynamic size, intrinsic disorder, and flexibility to the whole complex. Interestingly, these biophysical properties are partially mitigated by the presence of SpGemin8•SpGemin7•SpGemin6 subunits. These results classify the SpSMN complex as a multidomain entity connected with flexible linkers and characterize the SpSMN subunit to be the central oligomeric structural organizer of the whole complex.
The degradation of poly-adenosine tails of messenger RNAs (mRNAs) in the eukaryotic cells is a determining step in controlling the level of gene expression. The highly conserved Ccr4-Not complex was identified as the major deadenylation complex in all eukaryotic organisms. Plenty of biochemical studies have shown that this complex is also involved in many aspects of the mRNA metabolism, but we are still lacking the detailed structural information about its overall architecture and conformational states that could help to elucidate its multifunction and the way it is coordinated in the cells. Such information can also provide a basis to finding a possible way of intervention since the complex is also involved in some diseases such as cancer and cardiovascular disorders in humans. Meanwhile, the single particle Cryo-EM method has been through a “resolution revolution” recently due to the use of the newly developed direct electron detectors and has since resolved the high-resolution structures of many macromolecular protein complexes in their near-native state. Therefore, it was employed as a suitable method for studying the Ccr4-Not complex here. In this work, the Falcon 3EC direct detector mounted on the 300kV Titan Krios G3i Cryo-EM was evaluated for its practical performance at obtaining high-quality Cryo-EM data from protein samples of different molecular sizes. This served as a proof of principle for this detector’s capabilities and as a data collection guidance for studying the macromolecular complexes, such as the Ccr4-Not, when using an advanced high-performance microscope system. Next, the endogenous yeast Ccr4-Not complex was also purified via the immunoaffinity purification method and evaluated using negative staining EM to assess the conditions of the complex before proceeding to sample preparation for Cryo-EM. This has shown that the complex had an unexpected inherently dynamic property in vitro and extra optimisation procedures were needed to stabilise the complex during the purification and sample preparation. In addition, by using the label-free quantitative Mass spectrometry to examine the coimmunoprecipitated complex via different tagged subunits, it was deduced that two of the subunits (Not3/Not5) that shared some sequence similarity might compete for association with the scaffold subunit of the complex. An uncharacterised protein was also identified coimmunoprecipitating with the Caf130 subunit of the yeast complex. Cryo-EM data from the purified complex provided a low-resolution map that represents a surprisingly smaller partial complex as compared to 3D structures from previous studies, although gel electrophoresis and Mass spectrometry data have identified all of the nine subunits of the Ccr4-Not core complex in the sample. It was concluded that due to the presence of many predicted unstructured regions VI in the subunits and their dynamic composition in solution, the native complex could have been spontaneously denatured at the air/water interface during the sample preparation thus limiting the resolution of the Cryo-EM reconstruction. The purified complex was also examined for its deadenylase and ubiquitin ligase activity by in vitro assays. It was shown that the native complex has a different rate of activity and possibly also a different mode of action compared to the recombinant complexes from other species under similar reaction conditions. The Not4 E3 ligase was also shown to be active in the complex and was likely auto-ubiquitinated in the absence of a substrate. Both types of assays have also shown that the conformational flexibility does not seem to affect the enzymatic reactions when using a chemically crosslinked form of the complex for the assay, which implies that there can be other underlying mechanisms coordinating its structural and functional relationship. The findings from this work have therefore moved our understanding of the Ccr4-Not complex forward by looking at the different structural and functional behaviours of the endogenous complex, especially highlighting the obstacles in sample preparation for the native complex in high-resolution Cryo-EM. This would serve as foundation for future studies on the mechanism of this complex’s catalytic functions and also for optimising the Cryo-EM sample to generate better data that could eventually resolve the structure to a high-resolution.
The Atg12-Atg5/Atg16L1 complex is recruited by WIPI2b to the site of autophagosome formation. Atg16L1 is an effector of the Golgi resident GTPase Rab33B. Here we identified a minimal stable complex of murine Rab33B(30-202) Q92L and Atg16L1(153-210). Atg16L1(153-210) comprises the C-terminal part of the Atg16L1 coiled-coil domain. We have determined the crystal structure of the Rab33B Q92L/Atg16L1(153-210) effector complex at 3.47 angstrom resolution. This structure reveals that two Rab33B molecules bind to the diverging alpha -helices of the dimeric Atg16L1 coiled-coil domain. We mutated Atg16L1 and Rab33B interface residues and found that they disrupt complex formation in pull-down assays and cellular co-localization studies. The Rab33B binding site of Atg16L1 comprises 20 residues and immediately precedes the WIPI2b binding site. Rab33B mutations that abolish Atg16L binding also abrogate Rab33B association with the Golgi stacks. Atg16L1 mutants that are defective in Rab33B binding still co-localize with WIPI2b in vivo. The close proximity of the Rab33B and WIPI2b binding sites might facilitate the recruitment of Rab33B containing vesicles to provide a source of lipids during autophagosome biogenesis.
Background:
Pancreatic cancer is a fatal disease associated with resistance to conventional therapies. This study aimed to determine changes in gene expression patterns associated with infection and susceptibility of pancreatic cancer cells to an oncolyticvaccinia virus, GLV-1h153, carrying the human sodium iodide symporter for deep tissue imaging of virotherapy.
Methods:
Replication and susceptibility of pancreatic adenocarcinoma PANC-1 cells to GLV-1h153 was confirmed with replication and cytotoxicity assays. PANC-1 cells were then infected with GLV-1h153 and near-synchronous infection confirmed via flow cytometry of viral-induced green fluorescent protein (GFP) expression. Six and 24 hours after infection, three samples of each time point were harvested, and gene expression patterns assessed using HG-U133A cDNA microarray chips as compared to uninfected control. Differentially expressed genes were identified using Bioconductor LIMMA statistical analysis package. A fold change of 2.0 or above was used as a cutoff, with a P value of 0.01. The gene list was then analyzed using Ingenuity Pathways Analysis software.
Results:
Differential gene analysis revealed a total of 12,412 up- and 11,065 downregulated genes at 6 and 24 hours postinfection with GLV-1h153 as compared to control. At 6 hours postinfection. A total of 139 genes were either up or downregulated >twofold (false discovery rate < 0.05), of which 124 were mapped by Ingenuity Pathway Analysis (IPA). By 24 hours postinfection, a total of 5,698 genes were identified and 5,563 mapped by IPA. Microarray revealed gene expression changes, with gene networks demonstrating downregulation of processes such as cell death, cell cycle, and DNA repair, and upregulation of infection mechanisms (P < 0.01). Six hours after infection, gene changes involved pathways such as HMGB-1, interleukin (IL)-2, IL-6, IL-8, janus kinase/signal tranducer and activator of transcription (JAK/STAT), interferon, and ERK 5 signaling (P < 0.01). By 24 hours, prominent pathways included P53- and Myc-induced apoptotic processes, pancreatic adenocarcinoma signaling, and phosphoinositide 3-kinase/v-akt murine thymoma vial oncogene homolog 1 (PI3/AKT) pathways.
Conclusions:
Our study reveals the ability to assess time-dependent changes in gene expression patterns in pancreatic cancer cells associated with infection and susceptibility to vaccinia viruses. This suggests that molecular assays may be useful to develop safer and more efficacious oncolyticvirotherapies and support the idea that these treatments may target pathways implicated in pancreatic cancer resistance to conventional therapies.
The eukaryotic gene expression requires extensive regulations to enable the homeostasis of the cell and to allow dynamic responses due to external stimuli. Although many regulatory mechanisms involve the transcription as the first step of the gene expression, intensive regulation occurs also in the post-transcriptional mRNA metabolism. Thereby, the particular composition of the mRNPs plays a central role as the components associated with the mRNA form a specific “mRNP code” which determines the fate of the mRNA. Many proteins which are involved in this regulation and the mRNA metabolism are affected in diseases and especially neurological disorders often result from an aberrant mRNP code which leads to changes in the regulation and expression of mRNPs.
The focus of this work was on a trimeric protein complex which is termed TTF complex based on its subunits TDRD3, TOP3β and FMRP. Biochemical investigations revealed that the three components of the TTF complex are nucleo-cytosolic shuttle proteins which localize in the cytoplasm at the steady-state, associate with mRNPs and are presumably connected to the translation. Upon cellular stress conditions, the TTF components concentrate in stress granules. Thus, the TTF complex is part of the mRNP code, however its target RNAs and function are still completely unknown. Since the loss of functional FMRP results in the fragile X syndrome and TOP3β is associated with schizophrenia and intellectual disability, the TTF complex connects these phenotypically related neuro-psychiatric disorders with each other on a molecular level.
Therefore, the aim of this work was to biochemically characterize the TTF complex and to define its function in the mRNA metabolism. In this work, evidence was provided that TDRD3 acts as the central unit of the TTF complex and directly binds to FMRP as well as to TOP3β. Thereby, the interaction of TDRD3 and TOP3β is very stable, whereas FMRP is a dynamic component. Interestingly, the TTF complex is not bound directly to mRNA, but is recruited via the exon junction complex (EJC) to mRNPs. This interaction is mediated by a specific binding motif of TDRD3, the EBM. Upon biochemical and biological investigations, it was possible to identify the interactome of the TTF complex and to define the role in the mRNA metabolism. The data revealed that the TTF complex is mainly associated with “early” mRNPs and is probably involved in the pioneer round of translation. Furthermore, TOP3β was found to bind directly to the ribosome and thus, establishes a connection between the EJC and the translation machinery. A reduction of the TTF components resulted in selective changes in the proteome in cultured cells, whereby individual protein subsets seem to be regulated rather than the global protein expression.
Moreover, the enzymatic analysis of TOP3β indicated that TOP3β is a type IA topoisomerase which can catalytically attack not only DNA but also RNA. This aspect is particularly interesting with regard to the connection between early mRNPs and the translation which has been revealed in this work.
The data obtained in this work suggest that the TTF complex plays a role in regulating the metabolism of an early mRNP subset possibly in the course of the pioneer round of translation. Until now, the link between an RNA topoisomerase and the mRNA metabolism is thereby unique and thus provides a completely new perspective on the steps in the post-transcriptional gene expression and its regulation.