An N-Terminal Extension to UBA5 Adenylation Domain Boosts UFM1 Activation: Isoform-Specific Differences in Ubiquitin-like Protein Activation

Modification of proteins by the ubiquitin-like protein, UFM1, requires activation of UFM1 by the E1-activating enzyme, UBA5. In humans, UBA5 possesses two isoforms, each comprising an adenylation domain, but only one containing an N-terminal extension. Currently, the role of the N-terminal extension in UFM1 activation is not clear. Here we provide structural and biochemical data on UBA5 N-terminal extension to understand its contribution to UFM1 activation. The crystal structures of the UBA5 long isoform bound to ATP with and without UFM1 show that the N-terminus not only is directly involved in ATP binding but also affects how the adenylation domain interacts with ATP. Surprisingly, in the presence of the N-terminus, UBA5 no longer retains the 1:2 ratio of ATP to UBA5, but rather this becomes a 1:1 ratio. Accordingly, the N-terminus significantly increases the affinity of ATP to UBA5. Finally, the N-terminus, although not directly involved in the E2 binding, stimulates transfer of UFM1 from UBA5 to the E2, UFC1.     

Crystal Structure of the Human Ubiquitin-activating Enzyme 5 (UBA5) Bound to ATP: MECHANISTIC INSIGHTS INTO A MINIMALISTIC E1 ENZYME*

E1 ubiquitin-activating enzymes (UBAs) are large multidomain proteins that catalyze formation of a thioester bond between the terminal carboxylate of a ubiquitin or ubiquitin-like modifier (UBL) and a conserved cysteine in an E2 protein, producing reactive ubiquityl units for subsequent ligation to substrate lysines. Two important E1 reaction intermediates have been identified: a ubiquityl-adenylate phosphoester and a ubiquityl-enzyme thioester. However, the mechanism of thioester bond formation and its subsequent transfer to an E2 enzyme remains poorly understood. We have determined the crystal structure of the human UFM1 (ubiquitin-fold modifier 1) E1-activating enzyme UBA5, bound to ATP, revealing a structure that shares similarities with both large canonical E1 enzymes and smaller ancestral E1-like enzymes. In contrast to other E1 active site cysteines, which are in a variably sized domain that is separate and flexible relative to the adenylation domain, the catalytic cysteine of UBA5 (Cys²⁵⁰) is part of the adenylation domain in an α-helical motif. The novel position of the UBA5 catalytic cysteine and conformational changes associated with ATP binding provides insight into the possible mechanisms through which the ubiquityl-enzyme thioester is formed. These studies reveal structural features that further our understanding of the UBA5 enzyme reaction mechanism and provide insight into the evolution of ubiquitin activation.     

An atypical LIR motif within UBA5 (ubiquitin like modifier activating enzyme 5) interacts with GABARAP proteins and mediates membrane localization of UBA5

ABSTRACT

Short linear motifs, known as LC3-interacting regions (LIRs), interact with mactoautophagy/autophagy modifiers (Atg8/LC3/GABARAP proteins) via a conserved universal mechanism. Typically, this includes the occupancy of 2 hydrophobic pockets on the surface of Atg8-family proteins by 2 specific aromatic and hydrophobic residues within the LIR motifs. Here, we describe an alternative mechanism of Atg8-family protein interaction with the non-canonical UBA5 LIR, an E1-like enzyme of the ufmylation pathway that preferentially interacts with GABARAP but not LC3 proteins. By solving the structures of both GABARAP and GABARAPL2 in complex with the UBA5 LIR, we show that in addition to the binding to the 2 canonical hydrophobic pockets (HP1 and HP2), a conserved tryptophan residue N-terminal of the LIR core sequence binds into a novel hydrophobic pocket on the surface of GABARAP proteins, which we term HP0. This mode of action is unique for UBA5 and accompanied by large rearrangements of key residues including the side chains of the gate-keeping K46 and the adjacent K/R47 in GABARAP proteins. Swapping mutations in LC3B and GABARAPL2 revealed that K/R47 is the key residue in the specific binding of GABARAP proteins to UBA5, with synergetic contributions of the composition and dynamics of the loop L3. Finally, we elucidate the physiological relevance of the interaction and show that GABARAP proteins regulate the localization and function of UBA5 on the endoplasmic reticulum membrane in a lipidation-independent manner.

Abbreviations: ATG: AuTophaGy-related; EGFP: enhanced green fluorescent protein; GABARAP: GABA-type A receptor-associated protein; ITC: isothermal titration calorimetry; KO: knockout; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NMR: nuclear magnetic resonance; RMSD: root-mean-square deviation of atomic positions; TKO: triple knockout; UBA5: ubiquitin like modifier activating enzyme 5     

Structural basis for monoubiquitin recognition by the Ede1 UBA domain

Monoubiquitination is a general mechanism for downregulating the activity of cell surface receptors by consigning these proteins for lysosome-mediated degradation through the endocytic pathway. The yeast Ede1 protein functions at the internalization step of endocytosis and binds monoubiquitinated proteins through a ubiquitin associated (UBA) domain. UBA domains are found in a broad range of cellular proteins but previous studies have suggested that the mode of ubiquitin recognition might not be universally conserved. Here we present the solution structure of the Ede1 UBA domain in complex with monoubiquitin. The Ede1 UBA domain forms a three-helix bundle structure and binds ubiquitin through a largely hydrophobic surface in a manner reminiscent of the Dsk2 UBA and the remotely homologous Cue2 CUE domains, for which high-resolution structures have been described. However, the interaction is dissimilar to the molecular models proposed for the hHR23A UBA domains bound to either monoubiquitin or Lys48-linked diubiquitin. Our mutational analyses of the Ede1 UBA domain-ubiquitin interaction reveal several key affinity determinants and, unexpectedly, a negative affinity determinant in the wild-type Ede1 protein, implying that high-affinity interactions may not be the sole criterion for optimal function of monoubiquitin-binding endocytic proteins.     

Crystal structure of the ubiquitin-associated (UBA) domain of p62 and its interaction with ubiquitin

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.     

UBA domains of DNA damage-inducible proteins interact with ubiquitin

Rad23 is a highly conserved protein involved in nucleotide excision repair (NER) that associates with the proteasome via its N-terminus. Its C-terminal ubiquitin-associated (UBA) domain is evolutionarily conserved from yeast to humans. However, the cellular function of UBA domains is not completely understood. Recently, RAD23 and DDI1, both DNA damage-inducible genes encoding proteins with UBA domains, were implicated genetically in Pds1-dependent mitotic control in yeast. The UBA domains of RAD23 and DDI1 are required for these interactions. Timely degradation of Pds1 via the ubiquitin/proteasome pathway allows anaphase onset and is crucial for chromosome maintenance. Here, we show that Rad23 and Ddi1 interact directly with ubiquitin and that this interaction is dependent on their UBA domains, providing a possible mechanism for UBA-dependent cell cycle control. Moreover, we show that a hydrophobic surface on the UBA domain, which from structural work had been predicted to be a protein-protein interaction interface, is indeed required for ubiquitin binding. By demonstrating that UBA domains interact with ubiquitin, we have provided the first indication of a cellular function for the UBA domain.     

Atypical binding of the Swa2p UBA domain to ubiquitin

Swa2p is an auxilin-like yeast protein that is involved in vesicular transport and required for uncoating of clathrin-coated vesicles. Swa2p contains a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin (Ub)-mediated processes. We have determined a structural model of the Swa2p UBA domain in complex with Ub using NMR spectroscopy and molecular docking. Ub recognition occurs predominantly through an atypical interaction in which UBA helix α1 and the N-terminal part of helix α2 bind to Ub. Mutation of Ala148, a key residue in helix α1, to polar residues greatly reduced the affinity of the UBA domain for Ub and revealed a second low-affinity Ub-binding site located on the surface formed by helices α1 and α3. Surface plasmon resonance showed that the Swa2p UBA domain binds K48- and K63-linked di-Ub in a non-linkage-specific manner. These results reveal convergent evolution of a Ub-binding site on helix α1 of UBA domains involved in membrane protein trafficking.     

Impact of p62/SQSTM1 UBA domain mutations linked to Paget's disease of bone on ubiquitin recognition

The scaffold protein p62/SQSTM1 acts as a hub in regulating a diverse range of signaling pathways which are dependent upon a functional ubiquitin-binding C-terminal UBA domain. Mutations linked to Paget's disease of bone (PDB) commonly cluster within the UBA domain. The p62 UBA domain is unique in forming a highly stable dimer which regulates ubiquitin recognition by using overlapping surface patches in both dimerization and ubiquitin binding, making the two association events competitive. NMR structural analysis and biophysical methods show that some PDB mutations modulated the ubiquitin binding affinity by both direct and indirect mechanisms that affect UBA structural integrity, dimer stability, and contacts at the UBA-ubiquitin interface. In other cases, common PDB mutations (P392L in particular) result in no significant change in ubiquitin binding affinity for the UBA domain in isolation; however, all PDB UBA mutations lead to loss of function with respect to ubiquitin binding in the context of full-length p62, suggesting a more complex underlying mechanism.     

Proteins containing the UBA domain are able to bind to multi-ubiquitin chains

The UBA domain is a motif found in a variety of proteins, some of which are associated with the ubiquitin-proteasome system. We describe the isolation of a fission-yeast gene, mud1+, which encodes a UBA domain containing protein that is able to bind multi-ubiquitin chains. We show that the UBA domain is responsible for this activity. Two other proteins containing this motif, the fission-yeast homologues of Rad23 and Dsk2, are also shown to bind multi-ubiquitin chains via their UBA domains. These two proteins are implicated, along with the fission-yeast Pus1(S5a/Rpn10) subunit of the 26 S proteasome, in the recognition and turnover of substrates by this proteolytic complex.     

Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by sequestering lysine 48-linked polyubiquitin chains

Most substrates of the 26 S proteasome are recognized only following conjugation to a Lys48-linked polyubiquitin chain. Rad23 is one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA). Recent studies have shown that UbLs interact with 26 S proteasomes, whereas UBAs bind polyubiquitin chains. These biochemical properties suggest that UbL-UBA proteins may shuttle polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this model, the effect of human Rad23A on the degradation of polyubiquitinated substrates catalyzed by purified proteasomes is exclusively inhibitory. Strong inhibition is dependent on the presence of both UBAs, independent of the UbL, and can be explained by competition between the UBA domains and the proteasome for binding to substrate-linked polyubiquitin chains. The UBA domains bind Lys48-linked polyubiquitin chains in strong preference to Lys63 or Lys29-linked chains, leading to selective inhibition of the assembly and disassembly of Lys48-linked chains. These results place constraints on the mechanism(s) by which UbL-UBA proteins promote proteasome-catalyzed proteolysis and reveal new properties of UBA domains.     

蛋白质UFM化修饰

泛素折叠修饰因子1(ubiquitin-fold modifier 1,UFM1)是类泛素蛋白(ubiquitin-like modifier,UBL)家族的一员,存在于几乎所有的真核细胞中。UFM1对底物的修饰过程与泛素相似,即依次通过UBA5、UFC1和UFL1催化,共价接合在底物的赖氨酸残基上。而UFSP则负责切割UFM1的C端使之成熟,以及去除底物的UFM1修饰。UFM化修饰参与了内质网应激介导的细胞凋亡过程,对其具体作用机制的阐明需要鉴定到UFM1的修饰底物,但目前已经鉴定到UFM1的底物很少。大量研究尚聚焦于UFM修饰酶上。通过对UFM修饰酶和少量修饰底物的研究发现,UFM化修饰参与非酒精性肝病、细胞生成障碍性贫血、髋关节发育不良和神经系统疾病等的发生,以及乳腺癌细胞的增殖与转移和寄生虫的生长发育。本文将对UFM化修饰相关酶和修饰底物进行综述,总结UFM化修饰的生物学功能和在疾病发生发展中的作用。     

UBL/UBA ubiquitin receptor proteins bind a common tetraubiquitin chain

The ubiquitin-proteasome pathway is essential throughout the life cycle of a cell. This system employs an astounding number of proteins to ubiquitylate and to deliver protein substrates to the proteasome for their degradation. At the heart of this process is the large and growing family of ubiquitin receptor proteins. Within this family is an intensely studied group that contains both ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains: Rad23, Ddi1 and Dsk2. Although UBL/UBA family members are reported to regulate the degradation of other proteins, their individual roles in ubiquitin-mediated protein degradation has proven difficult to resolve due to their overlapping functional roles and interaction with each other and other ubiquitin family members. Here, we use a combination of NMR spectroscopy and molecular biology to reveal that Rad23 and Ddi1 interact with each other by using UBL/UBA domain interactions in a manner that does not preclude their interaction with ubiquitin. We demonstrate that UBL/UBA proteins can bind a common tetraubiquitin molecule and thereby provide strong evidence for a model in which chains adopt an opened structure to bind multiple receptor proteins. Altogether our results suggest a mechanism through which UBL/UBA proteins could protect chains from premature de-ubiquitylation and unnecessary elongation during their transit to the proteasome.     

UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme.

All known functions of ubiquitin are mediated through its covalent attachment to other proteins. The post-translational formation of ubiquitin--protein conjugates is preceded by an ATP-requiring step in which the carboxyl terminus of ubiquitin is adenylated and subsequently joined, through a thiolester bond, to a cysteine residue in the ubiquitin-activating enzyme, also known as E1. We report the isolation and functional analysis of the gene (UBA1) for the ubiquitin-activating enzyme of the yeast Saccharomyces cerevisiae. UBA1 encodes a 114 kd protein whose amino acid sequence contains motifs characteristic of nucleotide-binding sites. Expression of catalytically active UBA1 protein in E. coli, which lacks the ubiquitin system, confirmed that the yeast UBA1 gene encodes a ubiquitin-activating enzyme. Deletion of the UBA1 gene is lethal, demonstrating that the formation of ubiquitin--protein conjugates is essential for cell viability.     

UFMylation:A Unique & Fashionable Modification for Life

Ubiquitin-fold modifier 1 (UFM1) is one of the newly-identified ubiquitin-like proteins. Similar to ubiquitin, UFM1 is conjugated to its target proteins by a three-step enzymatic reaction. The UFM1-activating enzyme, ubiquitin-like modifier-activating enzyme 5 (UBA5), serves as the E1 to activate UFM1;UFM1-conjugating enzyme 1 (UFC1) acts as the E2 to transfer the activated UFM1 to the active site of the E2;and the UFM1-specific ligase 1 (UFL1) acts as the E3 to recog-nize its substrate, transfer, and ligate the UFM1 from E2 to the substrate. This process is called ufmylation. UFM1 chains can be cleaved from its target proteins by UFM1-specific proteases (UfSPs), suggesting that the ufmylation modification is reversible. UFM1 cascade is conserved among nearly all of the eukaryotic organisms, but not in yeast, and associated with several cellular activities including the endoplasmic reticulum stress response and hematopoiesis. Furthermore, the UFM1 cascade is closely related to a series of human diseases. In this review, we summarize the molecular details of this reversible modification process, the recent progress of its functional studies, as well as its implication in tumorigenesis and potential therapeutic targets for cancer.     

UBA1 and UBA2, two proteins that interact with UBP1, a multifunctional effector of pre-mRNA maturation in plants

Nicotiana plumbaginifolia UBP1 is an hnRNP-like protein associated with the poly(A)(+) RNA in the cell nucleus. Consistent with a role in pre-mRNA processing, overexpression of UBP1 in N. plumabaginifolia protoplasts enhances the splicing of suboptimal introns and increases the steady-state levels of reporter mRNAs, even intronless ones. The latter effect of UBP1 is promoter specific and appears to be due to UBP1 binding to the 3' untranslated region (3'-UTR) and protecting the mRNA from exonucleolytic degradation (M. H. L. Lambermon, G. G. Simpson, D. A. Kirk, M. Hemmings-Mieszczak, U. Klahre, and W. Filipowicz, EMBO J. 19:1638-1649, 2000). To gain more insight into UBP1 function in pre-mRNA maturation, we characterized proteins interacting with N. plumbaginifolia UBP1 and one of its Arabidopsis thaliana counterparts, AtUBP1b, by using yeast two-hybrid screens and in vitro pull-down assays. Two proteins, UBP1-associated proteins 1a and 2a (UBA1a and UBA2a, respectively), were identified in A. thaliana. They are members of two novel families of plant-specific proteins containing RNA recognition motif-type RNA-binding domains. UBA1a and UBA2a are nuclear proteins, and their recombinant forms bind RNA with a specificity for oligouridylates in vitro. As with UBP1, transient overexpression of UBA1a in protoplasts increases the steady-state levels of reporter mRNAs in a promoter-dependent manner. Similarly, overexpression of UBA2a increases the levels of reporter mRNAs, but this effect is promoter independent. Unlike UBP1, neither UBA1a nor UBA2a stimulates pre-mRNA splicing. These and other data suggest that UBP1, UBA1a, and UBA2a may act as components of a complex recognizing U-rich sequences in plant 3'-UTRs and contributing to the stabilization of mRNAs in the nucleus.     

Differential ubiquitin binding of the UBA domains from human c-Cbl and Cbl-b: NMR structural and biochemical insights

The Cbl proteins, RING-type E3 ubiquitin ligases, are responsible for ubiquitinating the activated tyrosine kinases and targeting them for degradation. Both c-Cbl and Cbl-b have a UBA (ubiquitin-associated) domain at their C-terminal ends, and these two UBA domains share a high sequence similarity (75%). However, only the UBA from Cbl-b, but not from c-Cbl, can bind ubiquitin (Ub). To understand the mechanism by which the UBA domains specifically interact with Ub with different affinities, we determined the solution NMR structures of these two UBA domains, cUBA from human c-Cbl and UBAb from Cbl-b. Their structures show that these two UBA domains share the same fold, a compact three-helix bundle, highly resembling the typical UBA fold. Chemical shift perturbation experiments reveal that the helix-1 and loop-1 of UBAb form a predominately hydrophobic surface for Ub binding. By comparing the Ub-interacting surface on UBAb and its counterpart on cUBA, we find that the hydrophobic patch on cUBA is interrupted by a negatively charged residue Glu12. Fluorescence titration data show that the Ala12Glu mutant of UBAb completely loses the ability to bind Ub, whereas the mutation disrupting the dimerization has no significant effect on Ub binding. This study provides structural and biochemical insights into the Ub binding specificities of the Cbl UBA domains, in which the hydrophobic surface distribution on the first helix plays crucial roles in their differential affinities for Ub binding. That is, the amino acid residue diversity in the helix-1 region, but not the dimerization, determines the abilities of various UBA domains binding with Ub.     

Ubiquitin-Associated (UBA) Domain in Human Fas Associated Factor 1 Inhibits Tumor Formation by Promoting Hsp70 Degradation

Human Fas associated factor 1 (hFAF1) is a pro-apoptotic scaffolding protein containing ubiquitin-associating (UBA), ubiquitin like 1 and 2 (UBL1, UBL2), and ubiquitin regulatory X (UBX) domains. hFAF1 interacts with polyubiquitinated proteins via its N-terminal UBA domain and with valosin containing protein (VCP) via its C-terminal UBX domain. Overexpression of hFAF1 or its N-terminal UBA domain significantly increases cell death by increasing the degradation of polyubiquitinated proteins. In this study, we investigated whether hFAF1, whose expression level is reduced in cervical cancer, plays a role in tumor formation. We found that HeLa cells overexpressing full-length hFAF1 or the hFAF1 UBA domain alone, significantly suppressed the anchorage independent tumor growth in soft agar colony formation, increased cell death, and activated JNK and caspase 3. Employing UBA-specific tandem immunoprecipitation, we identified moieties specifically interacting with UBA domain of hFAF1, and found that polyubiquitinated Hsp70s are recruited to UBA domain. We also demonstrated that hFAF1 overexpression promotes Hsp70 degradation via the proteasome. We further found that mutating the UBA domain (I41N), as well as knocking down hFAF1 with specific RNAi, abolishs its ability to increase the proteasomal degradation of Hsp70. These findings suggest that hFAF1 inhibits tumor formation by increasing the degradation of Hsp70 mediated via its UBA domain.     

Ubiquitin-activating enzyme UBA1 is required for cellular response to DNA damage

The cellular DNA damage response (DDR) machinery that maintains genomic integrity and prevents severe pathologies, including cancer, is orchestrated by signaling through protein modifications. Protein ubiquitylation regulates repair of DNA double-strand breaks (DSBs), toxic lesions caused by various metabolic as well as environmental insults such as ionizing radiation (IR). Whereas several components of the DSB-evoked ubiquitylation cascade have been identified, including RNF168 and BRCA1 ubiquitin ligases, whose genetic defects predispose to a syndrome mimicking ataxia-telangiectasia and cancer, respectively, the identity of the apical E1 enzyme involved in DDR has not been established. Here, we identify ubiquitin-activating enzyme UBA1 as the E1 enzyme required for responses to IR and replication stress in human cells. We show that siRNA-mediated knockdown of UBA1, but not of another UBA family member UBA6, impaired formation of both ubiquitin conjugates at the sites of DNA damage and IR-induced foci (IRIF) by the downstream components of the DSB response pathway, 53BP1 and BRCA1. Furthermore, chemical inhibition of UBA1 prevented IRIF formation and severely impaired DSB repair and formation of 53BP1 bodies in G₁, a marker of response to replication stress. In contrast, the upstream steps of DSB response, such as phosphorylation of histone H2AX and recruitment of MDC1, remained unaffected by UBA1 depletion. Overall, our data establish UBA1 as the apical enzyme critical for ubiquitylation-dependent signaling of both DSBs and replication stress in human cells, with implications for maintenance of genomic integrity, disease pathogenesis and cancer treatment.     

Modification of ERα by UFM1 Increases Its Stability and Transactivity for Breast Cancer Development

The post-translational modification (e.g., phosphorylation) of estrogen receptor α (ERα) plays a role in controlling the expression and subcellular localization of ERα as well as its sensitivity to hormone response. Here, we show that ERα is also modified by UFM1 and this modification (ufmylation) plays a crucial role in promoting the stability and transactivity of ERα, which in turn promotes breast cancer development. The elevation of ufmylation via the knockdown of UFSP2 (the UFM1-deconjugating enzyme in humans) dramatically increases ERα stability by inhibiting ubiquitination. In contrast, ERα stability is decreased by the prevention of ufmylation via the silencing of UBA5 (the UFM1-activating E1 enzyme). Lys171 and Lys180 of ERα were identified as the major UFM1 acceptor sites, and the replacement of both Lys residues by Arg (2KR mutation) markedly reduced ERα stability. Moreover, the 2KR mutation abrogated the 17β-estradiol-induced transactivity of ERα and the expression of its downstream target genes, including pS2, cyclin D1, and c-Myc; this indicates that ERα ufmylation is required for its transactivation function. In addition, the 2KR mutation prevented anchorage-independent colony formation by MCF7 cells. Most notably, the expression of UFM1 and its conjugating machinery (i.e., UBA5, UFC1, UFL1, and UFBP1) were dramatically upregulated in ERα-positive breast cancer cell lines and tissues. Collectively, these findings implicate a critical role attributed to ERα ufmylation in breast cancer development by ameliorating its stability and transactivity.     

GRP78 determines glioblastoma sensitivity to UBA1 inhibition-induced UPR signaling and cell death

Glioblastoma multiforme (GBM) is an extremely aggressive brain tumor for which new therapeutic approaches are urgently required. Unfolded protein response (UPR) plays an important role in the progression of GBM and is a promising target for developing novel therapeutic interventions. We identified ubiquitin-activating enzyme 1 (UBA1) inhibitor TAK-243 that can strongly induce UPR in GBM cells. In this study, we evaluated the functional activity and mechanism of TAK-243 in preclinical models of GBM. TAK-243 significantly inhibited the survival, proliferation, and colony formation of GBM cell lines and primary GBM cells. It also revealed a significant anti-tumor effect on a GBM PDX animal model and prolonged the survival time of tumor-bearing mice. Notably, TAK-243 more effectively inhibited the survival and self-renewal ability of glioblastoma stem cells (GSCs) than GBM cells. Importantly, we found that the expression level of GRP78 is a key factor in determining the sensitivity of differentiated GBM cells or GSCs to TAK-243. Mechanistically, UBA1 inhibition disrupts global protein ubiquitination in GBM cells, thereby inducing ER stress and UPR. UPR activates the PERK/ATF4 and IRE1α/XBP signaling axes. These findings indicate that UBA1 inhibition could be an attractive strategy that may be potentially used in the treatment of patients with GBM, and GRP78 can be used as a molecular marker for personalized treatment by targeting UBA1.Subject terms: CNS cancer, Cancer stem cells