Breaking Bad Proteins-Discovery Approaches and the Road to Clinic for Degraders.

Proteolysis-targeting chimeras (PROTACs) describe compounds that bind to and induce degradation of a target by simultaneously binding to a ubiquitin ligase. More generally referred to as bifunctional degraders, PROTACs have led the way in the field of targeted protein degradation (TPD), with several compounds currently undergoing clinical testing. Alongside bifunctional degraders, single-moiety compounds, or molecular glue degraders (MGDs), are increasingly being considered as a viable approach for development of therapeutics, driven by advances in rational discovery approaches.

SUMOylation controls Hu antigen R posttranscriptional activity in liver cancer.

The posttranslational modification of proteins critically influences many biological processes and is a key mechanism that regulates the function of the RNA-binding protein Hu antigen R (HuR), a hub in liver cancer. Here, we show that HuR is SUMOylated in the tumor sections of patients with hepatocellular carcinoma in contrast to the surrounding tissue, as well as in human cell line and mouse models of the disease. SUMOylation of HuR promotes major cancer hallmarks, namely proliferation and invasion, whereas the absence of HuR SUMOylation results in a senescent phenotype with dysfunctional mitochondria and endoplasmic reticulum.

SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells.

Background: The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown.

Methods: SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6-SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES).

LGG-1/GABARAP lipidation is not required for autophagy and development in .

The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy, such as initiation, cargo recognition and engulfment, vesicle closure and degradation. Most of LC3/GABARAP functions are considered dependent on their post-translational modifications and their association with the autophagosome membrane through a conjugation to a lipid, the phosphatidyl-ethanolamine. Contrarily to mammals, possesses single homologs of LC3 and GABARAP families, named LGG-2 and LGG-1.

A Computational Tool for Analysis of Mass Spectrometry Data of Ubiquitin-Enriched Samples.

Mass spectrometry data on ubiquitin and ubiquitin-like modifiers are becoming increasingly more accessible, and the coverage progressively deepen as methodologies mature. This type of mass spectrometry data is linked to specific data analysis pipelines for ubiquitin. This chapter describes a computational tool to facilitate analysis of mass spectrometry data obtained on ubiquitin-enriched samples.

Analysis of ATG8 Family Members Using LC3-Interacting Regions (LIR)-Based Molecular Traps.

The ATG8 family of proteins regulates the autophagy process from the autophagosome maturation and cargo recruitment up to degradation. Autophagy dysfunction is involved in the development of multiple diseases. The LC3 interacting region (LIR)-based molecular traps have been designed to isolate endogenous ATG8 proteins and their interactors in order to facilitate the study of selective autophagy events.

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.

Exploring selective autophagy events in multiple biologic models using LC3-interacting regions (LIR)-based molecular traps.

Autophagy is an essential cellular pathway that ensures degradation of a wide range of substrates including damaged organelles or large protein aggregates. Understanding how this proteolytic pathway is regulated would increase our comprehension on its role in cellular physiology and contribute to identify biomarkers or potential drug targets to develop more specific treatments for disease in which autophagy is dysregulated. Here, we report the development of molecular traps based in the tandem disposition of LC3-interacting regions (LIR).

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.

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).

Fluctuations in AKT and PTEN Activity Are Linked by the E3 Ubiquitin Ligase cCBL.

3-Poly-phosphoinositides (PIP) regulate cell survival, division, and migration. Both PI3-kinase (phosphoinositide-3-kinase) and PTEN (phosphatase and tensin-homolog in chromosome 10) control PIP levels, but the mechanisms connecting PI3-kinase and PTEN are unknown. Using non-transformed cells, the activation kinetics of PTEN and of the PIP-effector AKT were examined after the addition of growth factors.

SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes.

Development is orchestrated through a complex interplay of multiple transcription factors. The comprehension of this interplay will help us to understand developmental processes. Here we analyze the relationship between two key transcription factors: CBX4, a member of the Polycomb Repressive Complex 1 (PRC1), and SALL1, a member of the Spalt-like family with important roles in embryogenesis and limb development.

SUMOylation modulates the stability and function of PI3K-p110β.

Class I PI3K are heterodimers composed of a p85 regulatory subunit and a p110 catalytic subunit involved in multiple cellular functions. Recently, the catalytic subunit p110β has emerged as a class I PI3K isoform playing a major role in tumorigenesis. Understanding its regulation is crucial for the control of the PI3K pathway in p110β-driven cancers.

Proteome-wide identification of NEDD8 modification sites reveals distinct proteomes for canonical and atypical NEDDylation.

The ubiquitin-like molecule NEDD8 controls several biological processes and is a promising target for therapeutic intervention. NEDDylation occurs through specific NEDD8 enzymes (canonical) or enzymes of the ubiquitin system (atypical). Identification of NEDD8 sites on substrates is critical for delineating the processes controlled by NEDDylation.

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).

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.

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.

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer.

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases.

Identification of Small Molecules Disrupting the Ubiquitin Proteasome System in Malaria.

The ubiquitin proteasome system (UPS) is one of the main proteolytic pathways in eukaryotic cells, playing an essential role in key cellular processes such as cell cycling and signal transduction. Changes in some of the components of this pathway have been implicated in various conditions, including cancer and infectious diseases such as malaria. The success of therapies based on proteasome inhibitors has been shown in human clinical trials.

Regulation of the Ebola Virus VP24 Protein by SUMO.

Some viruses take advantage of conjugation of ubiquitin or ubiquitin-like proteins to enhance their own replication. One example is Ebola virus, which has evolved strategies to utilize these modification pathways to regulate the viral proteins VP40 and VP35 and to counteract the host defenses. Here, we show a novel mechanism by which Ebola virus exploits the ubiquitin and SUMO pathways.

Extra-cellular vesicles carry proteome of cancer hallmarks.

Through lateral transfer, extra-cellular vesicles (EVs) transport their DNA, miRNA, mRNA and proteins such as enzymes mediating drug resistance, transporters as well as growth factors to neighboring cells. By virtue of this horizontal transfer, EVs potentially regulate cell growth, migration, angiogenesis and metastasis and increase tissue permeability in cancer. Furthermore, EVs regulate immune factors and allow the tumor cells to evade immune recognition and cell death.

Using Ubiquitin Binders to Decipher the Ubiquitin Code.

Post-translational modifications (PTMs) by ubiquitin (Ub) are versatile, highly dynamic, and involved in nearly all aspects of eukaryote biological function. The reversibility and heterogeneity of Ub chains attached to protein substrates have complicated their isolation, quantification, and characterization. Strategies have emerged to isolate endogenous ubiquitylated targets, including technologies based on the use of Ub-binding peptides, such as tandem-repeated Ub-binding entities (TUBEs).

PROTEOSTASIS: A European Network to Break Barriers and Integrate Science on Protein Homeostasis.

Protein homeostasis (proteostasis) is at the core of cellular functions. The European network PROTEOSTASIS was created to steer research and foster collaborations in the interconnected fields of posttranslational modifications by ubiquitin family members and protein turnover by proteasome, autophagy, and lysosomal systems in health and diseases, across the kingdoms of life.

SUMOylation regulates LKB1 localization and its oncogenic activity in liver cancer.

Background: Even though liver kinase B1 (LKB1) is usually described as a tumor suppressor in a wide variety of tissues, it has been shown that LKB1 aberrant expression is associated with bad prognosis in Hepatocellular Carcinoma (HCC).

Methods: Herein we have overexpressed LKB1 in human hepatoma cells and by using histidine pull-down assay we have investigated the role of the hypoxia-related post-translational modification of Small Ubiquitin-related Modifier (SUMO)ylation in the regulation of LKB1 oncogenic role. Molecular modelling between LKB1 and its interactors, involved in regulation of LKB1 nucleocytoplasmic shuttling and LKB1 activity, was performed.

NEDDylation promotes nuclear protein aggregation and protects the Ubiquitin Proteasome System upon proteotoxic stress.

Spatial management of stress-induced protein aggregation is an integral part of the proteostasis network. Protein modification by the ubiquitin-like molecule NEDD8 increases upon proteotoxic stress and it is characterised by the formation of hybrid NEDD8/ubiquitin conjugates. However, the biological significance of this response is unclear.