ER-PHAGY ER-phagy: Selective degradation of the endoplasmic reticulum

ER-phagy involves selective degradation of endoplasmic reticulum (ER), which is dynamically remodelled to adapt its structure to cellular needs. We have previously shown that ER-phagy is regulated by a subset of reticulon-type receptors, namely FAM134B and RTN3, which was followed by the discovery of three additional ER-phagy receptors, Sec62, CCPG1 and TEX264. These ER-phagy receptors exhibit different preferences in localization at the specific ER subdomains, the cargo selectivity and remodeling of the ER architecture. Yet, the molecular mechanisms underlying ER-phagy receptor action, activation or selectivity for ER remodelling upon cellular stress, remain unexplored.

Our lab focuses on – (1) understanding the function and regulation of ER-phagy receptors and their binding partners, with particular interest in ER Chaperones; (2) understanding the underlying mechanisms of the delivery of portions of ER to the lysosome, mainly through identification of novel co-receptors required for structure support, sensing, scission and the delivery of ER fragments to the lysosome. To address these areas of interest, we are utilizing biochemical methods, mass spectrometry and CRISPR/Cas9 screening in collaboration with FCSC laboratory within our institute. We are utilizing mouse models to investigate the implications the impairment in ER-phagy pathway has in vivo and in a physiological context.

 

DNA DAMAGE DNA damage: SPRTN in control of DNA-repair, aging and cancer development

Spartan (SPRTN) is a mammalian metalloprotease that resolves DNA-protein crosslinks (DPCs), thus removing replication fork barriers and allowing cells to tolerate replication stress (Lopez-Mosqueda J et el., 2016). Mice with homozygous null alleles die very early during embryonic development. In humans, mutations in the SPRTN gene result in Ruijs-Aalfs syndrome, a segmental progeroid syndrome with an increased risk of hepatocellular carcinoma.

In our group, our aim is to investigate the mechanism of DPC repair by SPRTN in detail, as well as to understand the pathophysiology and molecular mechanism underlying cancer and aging-related morbidities. We attempt to achieve these goals by applying different cutting-edge technologies in structural biology, cell biology, proteomics and in vivo functional studies using mice mutants. We aim to translate such research discoveries into the development of novel small chemical molecules that can later be used to treat cancer and premature aging diseases.


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BACTERIA: Ubiquitinome during bacterial infection

Bacterial resistance to standard antibiotic treatment has evolved into a serious threat worldwide. We believe that deciphering pathogenic and host defense mechanisms during bacterial infections is the key to novel antibacterial strategies. Ubiquitin (Ub) drives innate immune receptor signaling as well as microbicidal programs including selective autophagy of intracellular pathogens (Gomes and Dikic, 2014). Although prokaryotes lack Ub-coding genes as well as the Ub-proteasome system, a wide range of bacterial pathogens acquired strategies to exploit the host Ub machinery for their life cycle, counteract host inflammatory signaling and immune defense programs. In our group, we intensively study Legionella- and Salmonella-induced cell response. We and others have demonstrated that intracellular ubiquitinated bacteria are recognized by various glycan-, Ub- and LC3-binding autophagy receptors including p62/SQSTM1, NDP52 and LGALS8/Galectin 8, OPTN and TAX1BP1, which all help to deliver the pathogen into autophagosomes for degradation (Wild et al., 2011, van Wijk et al., 2012, van Wijk et al., 2017). Using our established quantitative diGly proteomics platform, we recorded the first global ubiquitinome of host epithelial cells infected with Salmonella typhimurium (Fiskin et al, 2016). We further acquired knowledge of the Salmonella infection approaching other available in the lab techniques. The Legionella study focusses on dissecting the role of Ser-Ub.

Ser-UB Serine Ubiquitination: Finding Ub ligases, deubiquitinases and substrates
Legionella pneumophila is a gram negative bacteria that causes Legionnaires disease. Upon infection, the bacteria eject more than 300 effector proteins into the host, which then hijacks host cell metabolism for bacterial survival and proliferation in intracellular vacuoles (Legionella-containing vacuoles). One of the pathways that get reprogrammed by the bacteria is the cellular ubiquitination system. Legionella has a unique system of targeting the cellular ubiquitin pool by modifying ubiquitin by phosphoribosylation. This ubiquitin (PR-ubiquitin) is then transferred to host proteins by phosphoribose-linked serine ubiquitination.

Our lab focusses on dissecting the role of phosphoribose-linked serine ubiquitination using a combination of biochemistry, structural biology and mass spectrometry to discover the molecular basis of phosphoribose-linked ubiquitination catalyzed by SidE effector proteins. These are coupled to Legionella infection studies to understand the effect of bacterial effectors on cellular signaling pathways and organellar dynamics. PR-ubiquitination is a fine-tuned response and is regulated during infection using at least two other bacterial effectors – SidJ and Dups. From Cryo-EM studies and mass spectrometric signatures, we identified SidJ to be a bacterial glutamylase, which glutamylates and inactivates the active site of SidE proteins (Bhogaraju et al., 2019). Dups were also a recent serendipitous discovery and were characterized to be a group of deubiquitinases that can specifically reverse PR-ubiquitination (Shin et al., 2020). Using a catalytically dead Dup-trapping mutant, coupled to mass spectrometry, we have identified more than 180 cellular proteins that are modified by PR-ubiquitination during Legionella infection, showing a global effect of SidE effectors in reprogramming the host cell during infection.

 

VIRUS: Autophagy activation during RNA viral infection

Viruses are an intriguing system to study in terms of host-pathogen interaction. They can remodel host pathways for viral survival and replication. Enveloped positive-sense RNA viruses like coronaviruses have been a clinically important field of research as they cause frequent outbreaks affecting millions of people worldwide. These viruses are well-studied for their replication strategies and cellular entry pathways. However, how viral proteins hijack cellular signaling pathways remains a relatively unexplored area of research. We are currently studying Ub or UBL-specific proteases from coronaviruses like SARS, MERS and Covid-19 to see how these are similar and different from each other in terms of molecular structure, function and interaction with host cellular metabolism.

 

CANCER: Cancer biology

Cancer cells experience elevated levels of proteotoxic stress as a consequence of rapid growth, wide-spread missense mutations and genomic imbalance. Therefore, protein quality control systems, including the ubiquitin-proteasome system (UPS) and autophagy, become important to maintain the proteostasis of cancer cells. To this end, we are focusing our studies on decoding the roles of UPS system and autophagy in lung cancer, colorectal cancer and hepatocellular carcinoma. Several lines of evidence suggest that misregulation of components of the UPS (E3 ubiquitin ligases, deubiquitinases and E2s) contributes to the malignant progression of human cancers at multiple levels. Most of the ER-phagy receptors and proteins of the autophagy machinery have been linked to various malignancies, however, the molecular mechanistic insights governing these behaviors are poorly understood. By employing the state-of-the-art CRISPR/Cas screens, genetically engineered mouse models (GEMM) of cancer, and high-throughput quantitative mass spectrometry, we envision to decipher the key molecular signaling pathways responsible for the uncontrolled tumor growth, metastasis and the invasive potential. Lastly, based on this knowledge, we aim to develop novel therapeutic approaches to target specific cancer types.