Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics.

Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors.

Selective Modulation of the Human Glucocorticoid Receptor Compromises GR Chromatin Occupancy and Recruitment of p300/CBP and the Mediator Complex.

Exogenous glucocorticoids are frequently used to treat inflammatory disorders and as adjuncts for the treatment of solid cancers. However, their use is associated with severe side effects and therapy resistance. Novel glucocorticoid receptor (GR) ligands with a patient-validated reduced side effect profile have not yet reached the clinic.

A bispecific Clec9A-PD-L1 targeted type I interferon profoundly reshapes the tumor microenvironment towards an antitumor state.

Despite major improvements in immunotherapeutic strategies, the immunosuppressive tumor microenvironment remains a major obstacle for the induction of efficient antitumor responses. In this study, we show that local delivery of a bispecific Clec9A-PD-L1 targeted type I interferon (AcTaferon, AFN) overcomes this hurdle by reshaping the tumor immune landscape.Treatment with the bispecific AFN resulted in the presence of pro-immunogenic tumor-associated macrophages and neutrophils, increased motility and maturation profile of cDC1 and presence of inflammatory cDC2.

Targeting IFN activity to both B cells and plasmacytoid dendritic cells induces a robust tolerogenic response and protection against EAE.

Type I Interferon (IFN) was the very first drug approved for the treatment of Multiple Sclerosis (MS), and is still frequently used as a first line therapy. However, systemic IFN also causes considerable side effects, affecting therapy adherence and dose escalation. In addition, the mechanism of action of IFN in MS is multifactorial and still not completely understood.

Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly.

Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy.

Safe eradication of large established tumors using neovasculature-targeted tumor necrosis factor-based therapies.

Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity-on-Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF-based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo.

Targeting interferon activity to dendritic cells enables in vivo tolerization and protection against EAE in mice.

Type I Interferon (IFN) is widely used for multiple sclerosis (MS) treatment, but its side effects are limiting and its mechanism of action still unknown. Furthermore, 30-50% of MS patients are unresponsive, and IFN can even induce relapses. Fundamental understanding of the cellular target(s) of IFN will help to optimize treatments by reducing side effects and separating beneficial from detrimental effects.

Delivering Type I Interferon to Dendritic Cells Empowers Tumor Eradication and Immune Combination Treatments.

An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects.

Analyzing trapped protein complexes by Virotrap and SFINX.

The analysis of protein interaction networks is one of the key challenges in the study of biology. It connects genotypes to phenotypes, and disruption of such networks is associated with many pathologies. Virtually all the approaches to the study of protein complexes require cell lysis, a dramatic step that obliterates cellular integrity and profoundly affects protein interactions.

Trapping mammalian protein complexes in viral particles.

Cell lysis is an inevitable step in classical mass spectrometry-based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification.

Leptin's metabolic and immune functions can be uncoupled at the ligand/receptor interaction level.

The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. We here provide genetic and biochemical evidence that the metabolic and immune functions of leptin can be uncoupled at the receptor level. First, homozygous mutant fatt/fatt mice carry a spontaneous splice mutation causing deletion of the leptin receptor (LR) immunoglobulin-like domain (IGD) in all LR isoforms.

Selection of non-competitive leptin antagonists using a random nanobody-based approach.

The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. Accumulating evidence suggests that leptin plays a role in human pathologies, such as autoimmune diseases and cancer, thus providing a rationale for the development of leptin antagonists. In the present study, we generated and evaluated a panel of neutralizing nanobodies targeting the LR (leptin receptor).

MAPPIT (MAmmalian Protein-Protein Interaction Trap) as a tool to study HIV reverse transcriptase dimerization in intact human cells.

The high mutation rate of Human Immunodeficiency Virus (HIV) leads to the rapid derivation of compound-resistant virus strains and thus necessitates the identification and development of compounds with alternative mode of actions. MAPPIT (MAmmalian Protein-Protein Interaction Trap) is a highly efficient tool to study protein-protein interactions in intact human cells and is applied to study the dimerization process of the HIV reverse transcriptase complex. Highly specific signals for the p66/p51 and p66/p66 interactions could readily be detected.

HyperISGylation of Old World monkey ISG15 in human cells.

Background: ISG15 is an Ubiquitin-like protein, highly induced by Type I Interferons. Upon the cooperative activity of specific Ubiquitinating enzymes, ISG15 can be conjugated to its substrates. Increasing evidence points to a role for protein ISGylation in anti-viral and anti-tumoral defense.

Mammalian protein-protein interaction trap (MAPPIT) analysis of STAT5, CIS, and SOCS2 interactions with the growth hormone receptor.

Binding of GH to its receptor induces rapid phosphorylation of conserved tyrosine motifs that function as recruitment sites for downstream signaling molecules. Using mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, we mapped the binding sites in the GH receptor for signal transducer and activator of transcription 5 (STAT5) a and b and for the negative regulators of cytokine signaling cytokine-inducible Src-homology 2 (SH2)-containing protein (CIS) and suppressor of cytokine signaling 2 (SOCS2). Y534, Y566, and Y627 are the major recruitment sites for STAT5.

Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells.

Interactions between proteins are at the heart of the cellular machinery. It is therefore not surprising that altered interaction profiles caused by aberrant protein expression patterns or by the presence of mutations can trigger cellular dysfunction, eventually leading to disease. Moreover, many viral and bacterial pathogens rely on protein-protein interactions to exert their damaging effects.

Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter.

In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters. These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1+R1 and NBD2+R2.

Mapping of the leptin binding sites and design of a leptin antagonist.

The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III.

Design and use of a mammalian protein-protein interaction trap (MAPPIT).

Identifying the interaction partners of a protein is a straightforward way to gain insight into the protein's function and to position it in an interaction network such as a signal transduction pathway. Various techniques have been developed to serve this purpose, and some are specifically designed to study posttranslational modifications in mammalian proteins and to clarify their normal physiological context. However, several intrinsic constraints limit the use of these technologies, and most are not suitable for screening for new interacting partners.

Comparison of leptin- and interleukin-6-regulated expression of the rPAP gene family: evidence for differential co-regulatory signals.

The pancreatitis-associated protein (PAP)/regenerating protein (REG) family represents a complex group of small secretory proteins, which can function as acute phase reactants, lectins, antiapoptotic factors or growth factors for pancreatic beta-cells and neural cells. Transcriptional induction of rPAP/Reg genes was studied here in PC12 cells made responsive to leptin. Northern-blots showed quantitative differences in induction of four major family members by leptin and IL-6.