23rd Human Proteome Organization World Congress

Prof.

Assoc. Professor

Spatiotemporal dissection of protein localization to human primary cilia

Cell Biology Insights

Primary cilia are solitary, filamentous membrane protrusions emanating from the surface of most human cells. Most of what we know about their function stems from hereditary diseases that are caused by cilia dysfunction, so-called ciliopathies. Ciliopathy patients have a broad spectrum of phenotypes, underlining that cilia fulfil pleiotropic and tissue-specific functions. However, the molecular mechanisms underlying primary cilia function and cell-type specific roles of primary cilia remain poorly understood. This is mainly due to the lack of tools to reveal the protein composition of individual cilia. We address this challenge with antibody-based, spatial proteomics, and develop a cilia atlas as part of the subcellular section of the Human Protein Atlas (HPA) (https://www.proteinatlas.org/humanproteome/subcellular). We co-stained 1800 validated antibodies from the HPA with markers for nuclei, basal bodies, and primary cilia in three human cell lines of different tissue origin: hTERT-RPE1 cells from the retina, RPTEC/TERT1 cells from the kidney proximal tubules, and the mesenchymal stem cell line ASC52telo from the adipose tissue. We acquired high-resolution confocal 3D images and manually annotated as well as computationally analyzed each image.Our preliminary analysis revealed approximately 500 proteins to localize to primary cilia, with 200 of these proteins also detected at the basal body. We detected an additional 250 proteins that localized to basal bodies but not to cilia. The identified ciliary proteins highlight primary cilia as cellular antennae: At least 170 of the identified ciliary proteins are known to drive signal transduction and engage, e.g., in Hedgehog, GPCR, Calcium, TGFB, RTK, Rho GTPase, MAPK, or mTOR signaling. Furthermore, we observed a remarkable heterogeneity, both between cell lines and within supposedly identical cell populations. For approximately 75% of the identified ciliary proteins, we observed variable amounts in cilia of the same cell line. Furthermore, we identified only about 20% of the ciliary proteins in cilia of all three cell lines. Beyond that, for about 90% of the ciliary proteins, we observed co-localization to other subcellular compartments, with cytosol, basal body, microtubules of the cell body, and nucleoplasm being the most common other compartments. By computational analysis of the images, we relate the dynamic ciliary protein localization to cellular states.In conclusion, we reveal new ciliary proteins and demonstrate that the primary cilium is the most dynamic and diverse cellular compartment. Our cilia atlas will pave new avenues for basic and clinical research on cilia and ciliopathies.

Short talk

Professor

Prof

Diving into the hidden world of AltProts: unraveling functions and interactions

For several years, we have focused on identifying an extended proteome beyond the traditionally referenced proteins (RefProt) contained in databases. While most proteomic studies concentrate on these known proteins, there is another layer to the proteome called the "non-referenced or Ghost proteome," which includes "Alternative proteins" (AltProt). These proteins originate from regions typically annotated as non-coding in transcripts, such as the 5' and 3' untranslated regions (UTRs), alternative reading frames (+1 or +2), and non-coding RNAs (ncRNAs, lncRNAs, circRNAs). Because AltProts are not included in standard databases, they are difficult to detect, and their functions are not well understood. Our research aims to uncover these proteins and elucidate their roles in cellular processes.This raises the critical question: "How can we determine the function of AltProts given their lack of molecular tools?" To address this, we are developing innovative strategies to identify and characterize the functions of AltProts. By leveraging advanced techniques such as crosslinking mass spectrometry (XL-MS), we aim to reveal protein-protein interactions involving AltProts, thereby predicting their roles in various cellular pathways and processes. This approach has already shown promise, as we have identified interactions between AltProts and known proteins, providing insights into their potential functions and contributions to cellular mechanisms.Using the XL-MS approach, we can identify protein-protein interactions in an untargeted manner. This technique allows us to reveal the presence of AltProts within protein interaction networks and predict their roles in signaling pathways. For instance, using this approach, IP_166911.1 (AltATAD2) was found interacting with RPL10 and AUF1, possibly regulating the binding of 5S ribosomal RNA in the ribosome. Similarly, in glioblastoma cells (NCH82), several AltProts were identified interacting with TPM4, and some of these interactions were confirmed by Immunofluorescence and TurboID. We have optimized the detection of crosslinked peptides through subcellular fractionation techniques, enabling the identification of AltProts specific to cellular localization and their interactions. This approach revealed AltProts interacting with HLA-B, particularly at the B2M interaction site. AltProt IP_2284785 might be involved in the stability or inhibition of the MHC-I complex.Our ongoing research aims to expand the understanding of AltProts and their roles in cellular processes, potentially leading to new insights into cellular mechanisms and the development of novel therapeutic targets.

Professor Dr

A multi-proteomics approach identifies non-catalytic effects of ATP-competitive kinase inhibition

Structural Proteomics

Kinases are essential regulators of most cellular processes, and their activity is tightly controlled. Aberrant kinase function can contribute to the development and progression of various diseases. Small molecule inhibitors offer a means to correct this dysregulated kinase activity. Type I inhibitors are the largest group of kinase inhibitors and target the ATP-binding pocket of kinases in an ATP-competitive manner. While this mode of action has been well studied, it is largely unclear how these inhibitors could affect non-catalytic kinase functions by remodeling the structural dynamics and subsequent complex formation of kinases. Here we present a proteomics workflow based on affinity purification (AP) and limited proteolysis (LiP) coupled with mass spectrometry (MS) to probe changes in complex formation and kinase structure induced by these compounds. We show that inhibition with SGC-CAMKK2-1 induces CAMKK2 complex formation with known and candidate substrates, including the metabolic regulator AMPK, typically associated with activation of CAMKK2. To understand the mechanisms underlying inhibitor induced protein sequestration, we explored structural changes upon inhibition of CAMKK2 with the inhibitors SGC-CAMKK2-1 and staurosporine using AP-LiP-MS. Both inhibitors induced allosteric changes at the autoinhibitory domain, which is consistent with a relief of autoinhibition upon inhibitor binding. We propose that the inhibitor locks the kinase in a pseudoactive conformation that resembles the active, non-autoinhibited conformation, promoting stable complex formation with substrates and other proteins, potentially altering their activity. We observed similar structural changes upon inhibition of the related kinases CHEK1, DCLK1, CAMK1 and PKCα, suggesting a more general mechanism underlying Type I inhibition of kinases with autoinhibitory domains. We propose that holistic characterization of the effects of Type I inhibition can reveal crucial new insights into the molecular mechanisms that drive cellular phenotypes of existing and future kinase therapeutics beyond inhibition of kinase mediated substrate phosphorylation, to allow future expansion of the activities that could be targeted by ATP-competitive kinase inhibitors.

ECR session
Innovative approaches to study protein function

Appointment

Program

ECR sessionInnovative approaches to study protein function

Appointment

Program

ECR session
Innovative approaches to study protein function