Isolation and Mass Spectrometry Identification of K48 and K63 Ubiquitin Proteome Using Chain-Specific Nanobodies.

Protein ubiquitylation is an essential mechanism regulating almost all cellular functions in eukaryotes. The understanding of the role of distinct ubiquitin chains in different cellular processes is essential to identify biomarkers for disease diagnosis and prognosis but also to open new therapeutic possibilities. The high complexity of ubiquitin chains complicates this analysis, and multiple strategies have been developed over the last decades. Here, we report a protocol for the isolation and identification of K48 and K63 ubiquitin chains using chain-specific nanobodies associated to mass spectrometry. Different steps were optimized to increase the purification yield and reduce the binding on nonspecific proteins. The resulting protocol allows the enrichment of ubiquitin chain-specific targets from mammalian cells.

Constitutive Activation of p62/Sequestosome-1-Mediated Proteaphagy Regulates Proteolysis and Impairs Cell Death in Bortezomib-Resistant Mantle Cell Lymphoma.

Protein ubiquitylation coordinates crucial cellular events in physiological and pathological conditions. A comparative analysis of the ubiquitin proteome from bortezomib (BTZ)-sensitive and BTZ-resistant mantle cell lymphoma (MCL) revealed an enrichment of the autophagy-lysosome system (ALS) in BTZ-resistant cells. Pharmacological inhibition of autophagy at the level of lysosome-fusion revealed a constitutive activation of proteaphagy and accumulation of proteasome subunits within autophagosomes in different MCL cell lines with acquired or natural resistance to BTZ. Inhibition of the autophagy receptor p62/SQSTM1 upon verteporfin (VTP) treatment disrupted proteaphagosome assembly, reduced co-localization of proteasome subunits with autophagy markers and negatively impacted proteasome activity. Finally, the silencing or pharmacological inhibition of p62 restored the apoptosis threshold at physiological levels in BTZ-resistant cells both in vitro and in vivo. In total, these results demonstrate for the first time a proteolytic switch from the ubiquitin-proteasome system (UPS) to ALS in B-cell lymphoma refractory to proteasome inhibition, pointing out a crucial role for proteaphagy in this phenomenon and paving the way for the design of alternative therapeutic venues in treatment-resistant tumors.

Ubiquitin-chains dynamics and its role regulating crucial cellular processes.

The proteome adapts to multiple situations occurring along the life of the cell. To face these continuous changes, the cell uses posttranslational modifications (PTMs) to control the localization, association with multiple partners, stability, and activity of protein targets. One of the most dynamic protein involved in PTMs is Ubiquitin (Ub). Together with other members of the same family, known as Ubiquitin-like (UbL) proteins, Ub rebuilds the architecture of a protein in a few minutes to change its properties in a very efficient way. This capacity of Ub and UbL is in part due to their potential to form complex architectures when attached to target proteins or when forming Ub chains. The highly dynamic formation and remodeling of Ub chains is regulated by the action of conjugating and deconjugating enzymes that determine, in due time, the correct chain architecture for a particular cellular function. Chain remodeling occurs in response to physiologic stimuli but also in pathologic situations. Here, we illustrate well-documented cases of chain remodeling during DNA repair, activation of the NF-κB pathway and autophagy, as examples of this dynamic regulation. The crucial role of enzymes and cofactors regulating chain remodeling is discussed.

LUZP1 Controls Cell Division, Migration and Invasion Through Regulation of the Actin Cytoskeleton.

LUZP1 is a centrosomal and actin cytoskeleton-localizing protein that regulates both ciliogenesis and actin filament bundling. As the cytoskeleton and cilia are implicated in metastasis and tumor suppression, we examined roles for LUZP1 in the context of cancer. Here we show that LUZP1 exhibits frequent genomic aberrations in cancer, with a predominance of gene deletions. Furthermore, we demonstrate that CRISPR/Cas9-mediated loss of Luzp1 in mouse fibroblasts promotes cell migration and invasion features, reduces cell viability, and increases cell apoptosis, centriole numbers, and nuclear size while altering the actin cytoskeleton. Loss of Luzp1 also induced changes to ACTR3 (Actin Related Protein 3, also known as ARP3) and phospho-cofilin ratios, suggesting regulatory roles in actin polymerization, beyond its role in filament bundling. Our results point to an unprecedented role for LUZP1 in the regulation of cancer features through the control of actin cytoskeleton.

Inhibition of the proteasome and proteaphagy enhances apoptosis in FLT3-ITD-driven acute myeloid leukemia.

Acute myeloid leukaemia (AML) is a clonal disorder that affects hematopoietic stem cells or myeloid progenitors. One of the most common mutations that results in AML occurs in the gene encoding fms-like tyrosine kinase 3 (FLT3). Previous studies have demonstrated that AML cells expressing FLT3-internal tandem duplication (ITD) are more sensitive to the proteasome inhibitor bortezomib (Bz) than FLT3 wild-type cells, with this cytotoxicity being mediated by autophagy (Atg). Here, we show that proteasome inhibition with Bz results in modest but consistent proteaphagy in MOLM-14 leukemic cells expressing the FLT3-ITD mutation, but not in OCI-AML3 leukemic cells with wild-type FLT3. Chemical inhibition of Atg with bafilomycin A simultaneously blocked proteaphagy and resulted in the accumulation of the p62 Atg receptor in Bz-treated MOLM-14 cells. The use of ubiquitin traps revealed that ubiquitin plays an important role in proteasome-Atg cross-talk. The p62 inhibitor verteporfin blocked proteaphagy and, importantly, resulted in accumulation of high molecular weight forms of p62 and FLT3-ITD in Bz-treated MOLM-14 cells. Both Atg inhibitors enhanced Bz-induced apoptosis in FLT3-ITD-driven leukemic cells, highlighting the therapeutic potential of these treatments.

LUZP1, a novel regulator of primary cilia and the actin cytoskeleton, is a contributing factor in Townes-Brocks Syndrome.

Primary cilia are sensory organelles crucial for cell signaling during development and organ homeostasis. Cilia arise from centrosomes and their formation and function is governed by numerous factors. Through our studies on Townes-Brocks Syndrome (TBS), a rare disease linked to abnormal cilia formation in human fibroblasts, we uncovered the leucine-zipper protein LUZP1 as an interactor of truncated SALL1, a dominantly-acting protein causing the disease. Using TurboID proximity labeling and pulldowns, we show that LUZP1 associates with factors linked to centrosome and actin filaments. Here, we show that LUZP1 is a cilia regulator. It localizes around the centrioles and to actin cytoskeleton. Loss of LUZP1 reduces F-actin levels, facilitates ciliogenesis and alters Sonic Hedgehog signaling, pointing to a key role in cytoskeleton-cilia interdependency. Truncated SALL1 increases the ubiquitin proteasome-mediated degradation of LUZP1. Together with other factors, alterations in LUZP1 may be contributing to TBS etiology.

Primary cilia are the 'antennae' of animal cells: these small, flexible protrusions emerge from the surface of cells, where they help to sense and relay external signals. Cilia are assembled with the help of the cytoskeleton, a dynamic network of mesh-like filaments that spans the interior of the cell and controls many different biological processes. If cilia do not work properly, human diseases called ciliopathies can emerge. Townes-Brocks Syndrome (TBS) is an incurable disease that presents a range of symptoms such as malformations of the toes or fingers, hearing impairment, and kidney or heart problems. It is caused by a change in the gene that codes for a protein called SALL1, and recent work has also showed that the cells of TBS patients have defective cilia. In addition, this prior research identified a second protein that interacted with the mutant version of SALL1; called LUZP1, this protein is already known to help maintain the cytoskeleton. In this study, Bozal-Basterra et al. wanted to find out if LUZP1 caused the cilia defects in TBS. First, the protein was removed from mouse cells grown in the laboratory, which dramatically weakened the cytoskeleton. In keeping with this observation, both the number of cilia per cell and the length of the cilia were abnormal. Cells lacking LUZP1 also had defects in a signalling process that transmits signals received by cilia to different parts of the cell. All these defects were previously observed in cells isolated from TBS patients. In addition, LUZP1-deficient mouse cells showed the same problems with their cilia and cytoskeleton as the cells from individuals with TBS. Crucially, the cells from human TBS patients also had much lower levels of LUZP1 than normal, suggesting that the protein may contribute to the cilia defects present in this disease. The work by Bozal-Basterra et al. sheds light on how primary cilia depend on the cytoskeleton, while also providing new insight into TBS. In the future, this knowledge could help researchers to develop therapies for this rare and currently untreatable disease.

Mechanisms Regulating the UPS-ALS Crosstalk: The Role of Proteaphagy.

Protein degradation is tightly regulated inside cells because of its utmost importance for protein homeostasis (proteostasis). The two major intracellular proteolytic pathways are the ubiquitin-proteasome and the autophagy-lysosome systems which ensure the fate of proteins when modified by various members of the ubiquitin family. These pathways are tightly interconnected by receptors and cofactors that recognize distinct chain architectures to connect with either the proteasome or autophagy under distinct physiologic and pathologic situations. The degradation of proteasome by autophagy, known as proteaphagy, plays an important role in this crosstalk since it favours the activity of autophagy in the absence of fully active proteasomes. Recently described in several biological models, proteaphagy appears to help the cell to survive when proteostasis is broken by the absence of nutrients or the excess of proteins accumulated under various stress conditions. Emerging evidence indicates that proteaphagy could be permanently activated in some types of cancer or when chemoresistance is observed in patients.

Resistance to the Proteasome Inhibitors: Lessons from Multiple Myeloma and Mantle Cell Lymphoma.

Since its introduction in the clinics in early 2000s, the proteasome inhibitor bortezomib (BTZ) significantly improved the prognosis of patients with multiple myeloma (MM) and mantle cell lymphoma (MCL), two of the most challenging B cell malignancies in western countries. However, relapses following BTZ therapy are frequent, while primary resistance to this agent remains a major limitation for further development of its therapeutic potential. In the present chapter, we recapitulate the molecular mechanisms associated with intrinsic and acquired resistance to BTZ learning from MM and MCL experience, including mutations of crucial genes and activation of prosurvival signalling pathways inherent to malignant B cells. We also outline the preclinical and clinical evaluations of some potential druggable targets associated to BTZ resistance, considering the most meaningful findings of the past 10 years. Although our understanding of BTZ resistance is far from being completed, recent discoveries are contributing to develop new approaches to treat relapsed MM and MCL patients.

Truncated SALL1 Impedes Primary Cilia Function in Townes-Brocks Syndrome.

Townes-Brocks syndrome (TBS) is characterized by a spectrum of malformations in the digits, ears, and kidneys. These anomalies overlap those seen in a growing number of ciliopathies, which are genetic syndromes linked to defects in the formation or function of the primary cilia. TBS is caused by mutations in the gene encoding the transcriptional repressor SALL1 and is associated with the presence of a truncated protein that localizes to the cytoplasm. Here, we provide evidence that SALL1 mutations might cause TBS by means beyond its transcriptional capacity. By using proximity proteomics, we show that truncated SALL1 interacts with factors related to cilia function, including the negative regulators of ciliogenesis CCP110 and CEP97. This most likely contributes to more frequent cilia formation in TBS-derived fibroblasts, as well as in a CRISPR/Cas9-generated model cell line and in TBS-modeled mouse embryonic fibroblasts, than in wild-type controls. Furthermore, TBS-like cells show changes in cilia length and disassembly rates in combination with aberrant SHH signaling transduction. These findings support the hypothesis that aberrations in primary cilia and SHH signaling are contributing factors in TBS phenotypes, representing a paradigm shift in understanding TBS etiology. These results open possibilities for the treatment of TBS.