Single amino acid-based PROTACs trigger degradation of the oncogenic kinase BCR-ABL in chronic myeloid leukemia (CML).

Proteolysis-targeting chimera (PROTAC) that specifically targets harmful proteins for destruction by hijacking the ubiquitin-proteasome system is emerging as a potent anticancer strategy. How to efficiently modulate the target degradation remains a challenging issue. In this study, we employ a single amino acid-based PROTAC, which uses the shortest degradation signal sequence as the ligand of the N-end rule E3 ubiquitin ligases to degrade the fusion protein BCR (breakpoint cluster region)-ABL (Abelson proto-oncogene), an oncogenic kinase that drives the progression of chronic myeloid leukemia. We find that the reduction level of BCR-ABL can be easily adjusted by substituting different amino acids. Furthermore, a single PEG linker is found to achieve the best proteolytic effect. Our efforts have resulted in effective degradation of BCR-ABL protein by the N-end rule pathway and efficient growth inhibition of K562 cells expressing BCR-ABL in vitro and blunted tumor growth in a K562 xenograft tumor model in vivo. The PROTAC presented has unique advantages including lower effective concentration, smaller molecular size, and modular degradation rate. Demonstrating the efficacy of the N-end rule-based PROTACs in vitro and in vivo, our study further expands the limited degradation pathways currently available for PROTACs in vivo and is easily adapted for broader applications in targeted protein degradation.

The N-terminal domain of the non-receptor tyrosine kinase ABL confers protein instability and suppresses tumorigenesis.

Chromosome translocation can lead to chimeric proteins that may become oncogenic drivers. A classic example is the fusion of the BCR activator of RhoGEF and GTPase and the ABL proto-oncogene nonreceptor tyrosine kinase, a result of a chromosome abnormality (Philadelphia chromosome) that causes leukemia. To unravel the mechanism underlying BCR-ABL-mediated tumorigenesis, here we compared the stability of ABL and the BCR-ABL fusion. Using protein degradation, cell proliferation, 5-ethynyl-2-deoxyuridine, and apoptosis assays, along with xenograft tumor analysis, we found that the N-terminal segment of ABL, which is lost in the BCR-ABL fusion, confers degradation capacity that is promoted by SMAD-specific E3 ubiquitin protein ligase 1. We further demonstrate that the N-terminal deletion renders ABL more stable and stimulates cell growth and tumorigenesis. The findings of our study suggest that altered protein stability may contribute to chromosome translocation-induced cancer development.

A modular PROTAC design for target destruction using a degradation signal based on a single amino acid.

Proteolysis targeting chimeras (PROTACs) are bivalent molecules that bring a cellular protein to a ubiquitin ligase E3 for ubiquitination and subsequent degradation. Although PROTAC has emerged as a promising therapeutic means for cancers as it rewires the ubiquitin pathway to destroy key cancer regulators, the degradation signals/pathways for PROTACs remain underdeveloped. Here we append single amino acids, the simplest degradation signal, to a ligand specific for estrogen-related receptor α (ERRα) and demonstrate their utility in ERRα knockdown via the N-end rule pathway and also their efficiency in the growth inhibition of breast cancer cells. The modular design described offers unique advantages including smaller molecular size with shortest degradation sequences and degradation speed modulation with different amino acids. Our study expands the repertoire of limited ubiquitin pathways currently available for PROTACs and could be easily adapted for broad use in targeted protein degradation.

Autophagy regulator Atg9 is degraded by the proteasome.

Autophagy is a highly conserved biological process essential to protein, cellular and organismal homeostasis. As autophagy plays a critical role in cellular responses to various external and internal stimuli, it is important to understand the mechanism underlying autophagy regulation. Here, we monitor the stability of 17 key autophagy factors in the yeast S. cerevisiae and show that Atg9 and Atg14 are degraded under normal growth conditions. Whereas Atg14 is regulated by both the proteasome and autophagy, Atg9 turnover is normally mediated by the proteasome but impeded upon starvation or rapamycin treatment. Interestingly, distinct segments of Atg9 confer instability, suggesting that multiple pathways are involved in Atg9 degradation. Our results provide the foundation to further elucidate the physiological significance of Atg9 turnover and also the interplay between two major proteolytic systems (i.e., autophagy and the proteasome).

The F-box Protein Rcy1 Is Involved in the Degradation of Histone H3 Variant Cse4 and Genome Maintenance.

Cse4, a histone H3-like centromeric protein, plays critical functions in chromosome segregation. Cse4 level is tightly regulated, but the underlying mechanism remains poorly understood. We employed a toxicity-based screen to look for the degradation components involved in Cse4 regulation. Here, we show that the F-box containing protein Rcy1 is required for efficient Cse4 turnover as Cse4 degradation is compromised in yeast cells lacking RCY1 Excessive Cse4 accumulation in rcy1Δ cells leads to growth retardation. Furthermore, the deletion of RCY1 is tied to enhanced chromosome instability and temperature-sensitive cell growth. Our results reveal the involvement of Rcy1 in chromosome regulation and another regulatory pathway controlling the Cse4 level and activity.

Rad25 protein is targeted for degradation by the Ubc4-Ufd4 pathway.

Proteasome-mediated proteolysis provides dynamic spatial and temporal modulation of protein concentration in response to various intrinsic and extrinsic challenges. To gain a better understanding of the role of the proteasome in DNA repair, we systematically monitored the stability of 26 proteins involved in nucleotide excision repair (NER) under normal growth conditions. Among six NER factors found to be regulated by the proteasome, we further delineated the specific pathway involved in the degradation of Rad25, a subunit of TFIIH. We demonstrate that Rad25 turnover requires the ubiquitin-conjugating enzyme Ubc4 and the ubiquitin ligase Ufd4. Interestingly, the deletion of UFD4 specifically suppresses the rad25 mutant defective in transcription. Our results reveal a novel function of the Ufd4 pathway and another tie between the proteasome and NER regulators.

Heat shock protein 90ß stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells.

Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90ß (HSP90ß) interacts with FAK and the middle domain (amino acids 233-620) of HSP90ß is mainly responsible for this interaction. Furthermore, we found that HSP90ß regulates FAK stability since HSP90ß inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90ß interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90ß regulates FAK stability and identifies a potential therapeutic strategy to breast cancer.

The Cdc48 ATPase modulates the interaction between two proteolytic factors Ufd2 and Rad23.

Rad23 and cell division cycle protein 48 (Cdc48), two key regulators of postubiquitylation events, act on distinct and overlapping sets of substrates. The principle underlying their division of labor and cooperation in proteolysis remains elusive. Both Rad23 and Cdc48 bind a ubiquitin protein ligase ubiquitin fusion degradation-2 (Ufd2), and regulate the degradation of Ufd2 substrates. With its ability to bind ubiquitin chains directly and the proteasome via different domains, Rad23 serves as a bridge linking ubiquitylated substrates to the proteasome. The significance and specific role of the Ufd2-Cdc48 interaction are unclear. Here, we demonstrate that mutations in Ufd2 alter its interaction with Cdc48 and impair its function in substrate proteolysis but not in ubiquitylation. Furthermore, Cdc48 promotes the disassembly of the Ufd2-Rad23 complex in an manner that is dependent on ATP and Ufd2 binding, revealing a biochemical role for Cdc48. Rad23 was shown to bind separately to Ufd2 and to the proteasome subunit Rpn1, which define two distinct steps in proteolysis. The action of Cdc48 could free Rad23 from Ufd2 to allow its subsequent association with Rpn1, which in turn may facilitate the orderly transfer of the substrate from the ubiquitylation apparatus to the proteasome.

The Cdc48 protein and its cofactor Vms1 are involved in Cdc13 protein degradation.

Vms1 is a newly identified Cdc48-binding protein. The biological function of Vms1 remains obscure. Here, we show that both Cdc48 and Vms1, but not Cdc48 cofactors Ufd1 and Ufd2, are crucial for the degradation of Cdc13, a telomere regulator. Interestingly, both autophagy and the proteasome are involved in Cdc13 turnover. Toxicity associated with accumulation of large amounts of Cdc13 in vms1Δ or autophagy mutants underscores the significance of the proteolytic regulation of Cdc13. Because few ubiquitylated yeast proteins are known to be degraded by autophagy under non-stress conditions, the identification of Cdc13 as a target of autophagy provides a valuable tool to unravel the mechanism of autophagy-mediated selective protein degradation.

Ubiquitin ligase Ufd2 is required for efficient degradation of Mps1 kinase.

Ufd2 is a U-box-containing ubiquitylation enzyme that promotes ubiquitin chain assembly on substrates. The physiological function of Ufd2 remains poorly understood. Here, we show that ubiquitylation and degradation of the cell cycle kinase Mps1, a known target of the anaphase-promoting complex E3, require Ufd2 enzyme. Yeast cells lacking UFD2 exhibit altered chromosome stability and several spindle-related phenotypes, expanding the biological function of Ufd2. We demonstrate that Ufd2-mediated Mps1 degradation is conserved in humans. Our results underscore the significance of Ufd2 in proteolysis and further suggest that Ufd2-like enzymes regulate far more substrates than previously envisioned.

The PRMT5-LSD1 axis confers Slug dual transcriptional activities and promotes breast cancer progression.

BACKGROUND: Downregulation of epithelial markers and upregulation of mesenchymal markers are the characteristics of the epithelial to mesenchymal transition (EMT) program, which provides the metastatic advantage of breast cancer. However, the mechanism underlying the switch of EMT markers remains poorly understood. METHODS: In this study, we used the affinity purification and mass spectrometry coupled approach to identify the interactome of Slug. CoIP, GST-pulldown, ChIP, Re-ChIP, qPCR and Immunoblot were used to investigate the underlying mechanism of Slug-PRMT5-LSD1 complex. The role of PRMT5 and LSD1 in breast cancer progression was evaluated both in vivo and in vitro. RESULTS: Here we found that the transcription factor Slug associates with PRMT5 and LSD1 in a complex and facilitates the breast cancer invasion in vitro. Mechanistically, PRMT5 and LSD1 work with Slug to exert dual transcriptional activities to inhibit E-cadherin expression by PRMT5-catalyzed H4R3me2s and LSD1-mediated demethylation of H3K4me2 on the E-cadherin (CDH1) promoter, and activate vimentin (VIM) expression via PRMT5-driven H3R2me2s and LSD1-mediated removal of H3K9me2. Importantly, PRMT5 and LSD1 are coordinately expressed in breast cancer patients and pharmacologic perturbation of both PRMT5 and LSD1 shows a synergetic effect on the inhibition of breast tumor growth and metastasis in vivo. CONCLUSIONS: Our study suggests that PRMT5 and LSD1 function as a dual epigenetic modifier to promote Slug induced EMT program, suggesting that the inhibition of PRMT5 and LSD1 presents a potential therapeutic strategy against cancer metastasis.

How many authors does it take to publish a high profile or classic paper?

Although the process of publishing a scientific paper has gotten simpler, it is increasingly difficult to publish a paper in high profile journals. We have analyzed the publishing data in the cell biology field and found several alarming trends developing over the last two decades. There is an emerging divide between scientist-run journals and professional-run high profile journals. How did this happen? What should we do? The core issue is whether the current standard for high profile journals hurts rather than helps the scientific discovery process. In this regard, we suggest that the editors and scientists should direct their focus on the potential impact and rigor of the work instead of the "perfection" or "completeness" of the study.

Identification of an Htm1 (EDEM)-dependent, Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) pathway in Saccharomyces cerevisiae: application of a novel assay for glycoprotein ERAD.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related alpha-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.

Ubiquitin chain elongation enzyme Ufd2 regulates a subset of Doa10 substrates.

Ufd2 is the founding member of E4 enzymes that are specifically involved in ubiquitin chain elongation but whose roles in proteolysis remain scarce. Here, using a genome-wide screen, we identified one cellular target of yeast Ufd2 as the membrane protein Pex29. The ubiquitin chains assembled on Pex29 in vivo by Ufd2 mainly contain Lys-48 linkages. We found that the ubiquitin-protein E3 ligase for overexpressed Pex29 is Doa10, which is known to be involved in protein quality control. Interestingly, not all Doa10 substrates are regulated by Ufd2, suggesting that E4 involvement is not specific to a particular E3, but may depend on the spatial arrangement of the E3-substrate interaction. Cells lacking UFD2 elicit an unfolded protein response, expanding the physiological function of Ufd2. Our results lead to novel insights into the biological role of Ufd2 and further underscore the significance of Ufd2 in proteolysis.

The Png1-Rad23 complex regulates glycoprotein turnover.

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1-Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.

PCNA Ubiquitylation: Instructive or Permissive to DNA Damage Tolerance Pathways?

DNA lesions escaping from repair often block the DNA replicative polymerases required for DNA replication and are handled during the S/G2 phases by the DNA damage tolerance (DDT) mechanisms, which include the error-prone translesion synthesis (TLS) and the error-free template switching (TS) pathways. Where the mono-ubiquitylation of PCNA K164 is critical for TLS, the poly-ubiquitylation of the same residue is obligatory for TS. However, it is not known how cells divide the labor between TLS and TS. Due to the fact that the type of DNA lesion significantly influences the TLS and TS choice, we propose that, instead of altering the ratio between the mono- and poly-Ub forms of PCNA, the competition between TLS and TS would automatically determine the selection between the two pathways. Future studies, especially the single integrated lesion "i-Damage" system, would elucidate detailed mechanisms governing the choices of specific DDT pathways.

Circulating Tumor Cells (CTCs): A Unique Model of Cancer Metastases and Non-invasive Biomarkers of Therapeutic Response.

Late-stage cancer metastasis remains incurable in the clinic and is the major cause death in patients. Circulating tumor cells (CTCs) are thought to be metastatic precursors shed from the primary tumor or metastatic deposits and circulate in the blood. The molecular network regulating CTC survival, extravasation, and colonization in distant metastatic sites is poorly defined, largely due to challenges in isolating rare CTCs. Recent advances in CTC isolation and ex vivo culture techniques facilitates single-cell omics and the development of related animal models to study CTC-mediated metastatic progression. With these powerful tools, CTCs can potentially be used as non-invasive biomarkers predicting therapeutic response. These studies may open a new avenue for CTC-specific drug discoveries. In this short review, we aim to summarize recent progress in the characterization of CTCs and their clinical relevance in various cancers, setting the stage for realizing personalized therapies against metastases.

Positive feedback of SuFu negating protein 1 on Hedgehog signaling promotes colorectal tumor growth.

Hedgehog (Hh) signaling plays a critical role in embryogenesis and tissue homeostasis, and its deregulation has been associated with tumor growth. The tumor suppressor SuFu inhibits Hh signaling by preventing the nuclear translocation of Gli and suppressing cell proliferation. Regulation of SuFu activity and stability is key to controlling Hh signaling. Here, we unveil SuFu Negating Protein 1 (SNEP1) as a novel Hh target, that enhances the ubiquitination and proteasomal degradation of SuFu and thus promotes Hh signaling. We further show that the E3 ubiquitin ligase LNX1 plays a critical role in the SNEP1-mediated degradation of SuFu. Accordingly, SNEP1 promotes colorectal cancer (CRC) cell proliferation and tumor growth. High levels of SNEP1 are detected in CRC tissues and are well correlated with poor prognosis in CRC patients. Moreover, SNEP1 overexpression reduces sensitivity to anti-Hh inhibitor in CRC cells. Altogether, our findings demonstrate that SNEP1 acts as a novel feedback regulator of Hh signaling by destabilizing SuFu and promoting tumor growth and anti-Hh resistance.

A genome-wide synthetic dosage lethality screen reveals multiple pathways that require the functioning of ubiquitin-binding proteins Rad23 and Dsk2.

BACKGROUND: Ubiquitin regulates a myriad of important cellular processes through covalent attachment to its substrates. A classic role for ubiquitin is to flag proteins for destruction by the proteasome. Recent studies indicate that ubiquitin-binding proteins (e.g. Rad23, Dsk2, Rpn10) play a pivotal role in transferring ubiquitylated proteins to the proteasome. However, the specific role of these ubiquitin receptors remains poorly defined. A key to unraveling the functions of these ubiquitin receptors is to identify their cellular substrates and biological circuits they are involved in. Although many strategies have been developed for substrate isolation, the identification of physiological targets of proteolytic pathways has proven to be quite challenging. RESULTS: Using a genome-wide functional screen, we have identified 11 yeast genes that cause slower growth upon their overexpression in cells lacking two ubiquitin-binding proteins Rad23 and Dsk2. Our results suggest that proper functioning of Rad23 and Dsk2 is required for efficient pheromone response, transcription, amino acid metabolism, and DNA damage response. Two proteins identified by the screen are shown to be proteolytic substrates of Dsk2, validating the large scale synthetic dosage lethality screen as a new strategy for identifying substrates of a specific degradation pathway. CONCLUSION: In conclusion, as proof-of-concept, we show that a synthetic dosage lethality screen, which is based on the toxicity induced by gene overexpression, offers an effective, complementary method to elucidating biological functions of proteolytic pathways.

A Simple PCR-based Strategy for the Introduction of Point Mutations in the Yeast Saccharomyces cerevisiae via CRISPR/Cas9.

The methods currently employed for in vivo site-directed mutagenesis in yeast are laborious and/or inefficient. Recent developments of the CRISPR-based approaches hold great promise for genome editing, but its application in the yeast S. cerevisiae remains a time-consuming affair. The rate-limiting step in CRISPR-mediated genetic engineering in yeast is the incorporation of the guide sequences, which target Cas9 to relevant chromosomal locus, into the relevant yeast vectors. Here we present a PCR-based strategy to introduce specific point mutation into the yeast CDC48 gene via CRISPR. Our method eliminates the need for special dam- strain and markedly shortens the elaborate multi-step cloning process, leading to significant savings in time, labor and cost.