Human Homologue Research Articles

Mechanistic insights into nucleosomal H2B monoubiquitylation mediated by yeast Bre1-Rad6 and its human homolog RNF20/RNF40-hRAD6A

Histone H2B monoubiquitylation plays essential roles in chromatin-based transcriptional processes. A RING-type E3 ligase (yeast Bre1 or human RNF20/RNF40) and an E2 ubiquitin-conjugating enzyme (yeast Rad6 or human hRAD6A), together, precisely deposit ubiquitin on H2B K123 in yeast or K120 in humans. Here, we developed a chemical trapping strategy and successfully captured the transient structures of Bre1- or RNF20/RNF40-mediated ubiquitin transfer from Rad6 or hRAD6A to nucleosomal H2B. Our structures show that Bre1 and RNF40 directly bind nucleosomal DNA, exhibiting a conserved E3/E2/nucleosome interaction pattern from yeast to humans for H2B monoubiquitylation. We also find an uncanonical non-hydrophobic contact in the Bre1 RING-Rad6 interface, which positions Rad6 directly above the target H2B lysine residue. Our study provides mechanistic insights into the site-specific monoubiquitylation of H2B, reveals a critical role of nucleosomal DNA in mediating E3 ligase recognition, and provides a framework for understanding the cancer-driving mutations of RNF20/RNF40.

Phenothiazine-based virtual screening, molecular docking, and molecular dynamics of new trypanothione reductase inhibitors of Trypanosoma cruzi.

Phenothiazine derivatives can unselectively inhibit the trypanothione-dependent antioxidant system enzyme trypanothione reductase (TR). A virtual screening of 2163 phenothiazine derivatives from the ZINC15 and PubChem databases docked on the active site of T. cruzi TR showed that 285 compounds have higher affinity than the natural ligand trypanothione disulfide. 244 compounds showed higher affinity toward the parasite's enzyme than to its human homolog glutathione reductase. Protein-ligand interaction profiling predicted that the main interactions for the top scored compounds were with residues important for trypanothione disulfide binding: Phe396, Pro398, Leu399, His461, Glu466, and Glu467, particularly His461, which participates in catalysis. Two compounds with the desired profiles, ZINC1033681 (Zn_C687) and ZINC10213096 (Zn_C216), decreased parasite growth by 20 % and 50 %, respectively. They behaved as mixed-type inhibitors of recombinant TR, with Ki values of 59 and 47 μM, respectively. This study provides a further understanding of the potential of phenothiazine derivatives as TR inhibitors.

The yeast dynamin-like GTPase Vps1 mediates Atg9 transport to the phagophore assembly site in Saccharomyces cerevisiae.

Macroautophagy/autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis in eukaryotes. During autophagy, cisternal compartments called phagophores are generated to sequester intracellular components; these structures mature into autophagosomes, which deliver the cargo into lysosomes/vacuoles for degradation. Numerous autophagy-related (Atg) proteins are part of the core machinery that mediates autophagosome biogenesis. Atg9, a lipid scramblase and the only multispanning transmembrane protein among the core Atg machinery, traffics between cytoplasmic reservoirs and the phagophore assembly site (PAS) to provide membranes, recruit other Atg proteins and rearrange lipids on the phagophore membrane. However, the factors mediating Atg9 trafficking remain to be fully understood. In our recent study, we found that the yeast dynamin-like GTPase Vps1 (vacuolar protein sorting 1) is involved in autophagy and is important for Atg9 transport to the PAS. Moreover, we showed that Vps1 function in autophagy requires its GTPase and oligomerization activities. Interestingly, specific mutations in DNM2 (dynamin 2), one of the human homologs of Vps1 that have been linked with specific human diseases such as microcytic anemia and Charcot-Marie-Tooth, also impairs Atg9 transport to the PAS, suggesting that a defect in autophagy may underlay the pathophysiology of these severe human pathologies.

A Msp1-containing complex removes orphaned proteins in the mitochondrial outer membrane of T. brucei.

The AAA-ATPase Msp1 extracts mislocalised outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pulldown experiments, we show that trypanosomal Msp1 localises to both glycosomes and the mitochondrial outer membrane, where it forms a complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of α-helically anchored mitochondrial outer membrane proteins such as protein translocase subunits. Inhibition of their assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double, and triple RNAi cell lines combined with proteomic analyses, we demonstrate that not only Msp1 but also the trypanosomal homolog of the AAA-ATPase VCP are implicated in this quality control pathway. Moreover, in the absence of VCP three out of the four Msp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates, suggesting they act in concert with Msp1. pATOM36 is a functional analog of the yeast mitochondrial import complex complex and possibly of human mitochondrial animal-specific carrier homolog 2, suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans.

Understanding the Human RECQ5 Helicase-Connecting the Dots from DNA to Clinics.

RECQ5, a member of the conserved RECQ helicase family, is the sole human RECQ homolog that has not been linked to a hereditary developmental syndrome. Nonetheless, dysregulation of RECQ5 has emerged as a significant clinical concern, being linked to cancer predisposition, cardiovascular disease, and inflammation. In cells, RECQ5 assumes a crucial role in the regulation of DNA repair pathways, particularly in the repair of DNA double-strand breaks and inter-strand DNA crosslinks. Moreover, RECQ5 exhibits a capacity to modulate gene expression by interacting with transcription machineries and their co-regulatory proteins, thus safeguarding against transcription-induced DNA damage. This review aims to provide an overview of the multifaceted functions of RECQ5 and its implications in maintaining genomic stability. We will discuss the potential effects of clinical variants of RECQ5 on its cellular functions and their underlying mechanisms in the pathogenesis of cancer and cardiovascular disease. We will review the impact of RECQ5 variants in the field of pharmacogenomics, specifically their influence on drug responses, which may pave the way for novel therapeutic interventions targeting RECQ5 in human diseases.

MTH1 protects platelet mitochondria from oxidative damage and regulates platelet function and thrombosis

Human MutT Homolog 1 (MTH1) is a nucleotide pool sanitization enzyme that hydrolyzes oxidized nucleotides to prevent their mis-incorporation into DNA under oxidative stress. Expression and functional roles of MTH1 in platelets are not known. Here, we show MTH1 expression in platelets and its deficiency impairs hemostasis and arterial/venous thrombosis in vivo. MTH1 deficiency reduced platelet aggregation, phosphatidylserine exposure and calcium mobilization induced by thrombin but not by collagen-related peptide (CRP) along with decreased mitochondrial ATP production. Thrombin but not CRP induced Ca2+-dependent mitochondria reactive oxygen species generation. Mechanistically, MTH1 deficiency caused mitochondrial DNA oxidative damage and reduced the expression of cytochrome c oxidase 1. Furthermore, MTH1 exerts a similar role in human platelet function. Our study suggests that MTH1 exerts a protective function against oxidative stress in platelets and indicates that MTH1 could be a potential therapeutic target for the prevention of thrombotic diseases.

Structure-Guided Discovery of N5-CAIR Mutase Inhibitors.

Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N5-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N5-CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC50 values. Several of these compounds, including the α1-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.

MIF-like domain containing protein orchestrates cellular differentiation and virulence in the fungal pathogen Magnaporthe oryzae

Macrophage migration inhibitory factor (MIF) is a pleiotropic protein with chemotactic, pro-inflammatory, and growth-promoting activities first discovered in mammals. In parasites, MIF homologs are involved in immune evasion and pathogenesis. Here, we present the first comprehensive analysis of an MIF protein from the devastating plant pathogen Magnaporthe oryzae (Mo). The fungal genome encodes a single MIF protein (MoMIF1) that, unlike the human homolog, harbors multiple low-complexity regions (LCRs) and is unique to Ascomycota. Following infection, MoMIF1 is expressed in the biotrophic phase of the fungus, and is strongly down-regulated during subsequent necrotrophic growth in leaves and roots. We show that MoMIF1 is secreted during plant infection, affects the production of the mycotoxin tenuazonic acid and inhibits plant cell death. Our results suggest that MoMIF1 is a novel key regulator of fungal virulence that maintains the balance between biotrophy and necrotrophy during the different phases of fungal infection.

Abstract GS.102: Cd206 + Il-4rα + Macrophages Are Drivers Of Adverse Cardiac Remodeling In Ischemic Cardiomyopathy

Background: Cardiac macrophages are significantly expanded in chronic heart failure (HF); however, their role is unclear. We tested the hypothesis that CD206 + macrophages expressing interleukin (IL)-4Rα in the failing heart are key drivers of adverse left ventricular (LV) remodeling and ischemic cardiomyopathy. Methods and Results: Adult male C57BL/6 (WT) mice underwent non-reperfused myocardial infarction (MI) to induce HF, or sham operation, and cardiac macrophages were profiled using flow cytometry. As compared to sham hearts, both the frequency and absolute number of CD206 + macrophages steadily increased in post-MI hearts during the progression of LV remodeling and HF, such that at 8 w post-MI they comprised ~85% of macrophages. These macrophages were exclusively proliferative and predominantly C-C motif chemokine receptor (CCR2) - with nearly half expressing IL-4Rα, and correlated with LV dysfunction and fibrosis. IL-4-polarized bone marrow derived CD206 + macrophages exhibited marked upregulation of found in inflammatory zone (FIZZ)1 and induced FIZZ1-dependent myofibroblast differentiation of cardiac mesenchymal stem cells (cMSCs), in part related to DLL-4/Jagged1-Notch1 signaling. Intramyocardial adoptive transfer of M[IL-4], but not M[IL-10], macrophages induced progressive LV dilation and dysfunction and augmented cardiac fibrosis over 4 weeks in naïve mice. In vivo IL-4Rα gene silencing in mice with established HF effectively depleted cardiac CD206 + IL-4Rα + macrophages and induced reverse LV remodeling, improving fibrosis, neovascularization, and dysfunction, and suppressing local and systemic inflammation. Lastly, alternatively activated CD206 + and CD163 + macrophages were significantly expanded in human failing hearts and correlated with cardiac fibrosis, with the majority of CD163 + macrophages expressing IL-4Rα and FIZZ3, the human homolog of FIZZ1. Conclusion: CD206 + IL-4Rα + macrophages proliferate and expand in the failing heart and are key mediators of pathological remodeling and fibrosis, in part through the secretion of FIZZ1. Inhibition of CD206 + macrophage IL-4Rα signaling may be a fruitful therapeutic approach for alleviating LV remodeling in HF.

E4 ubiquitin ligase promotes mitofusin turnover and mitochondrial stress response

Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate cellular stress through their ubiquitylation, which is carried out by multiple E3 enzymes in response to many different stimuli. However, the molecular mechanisms that enable coordinated responses are largely unknown. Here we show that yeast Ufd2, a conserved ubiquitin chain-elongating E4 enzyme, is required for mitochondrial shape adjustments. Under various stresses, Ufd2 translocates to mitochondria and triggers mitofusin ubiquitylation. This elongates ubiquitin chains on mitofusin and promotes its proteasomal degradation, leading to mitochondrial fragmentation. Ufd2 and its human homologue UBE4B also target mitofusin mutants associated with Charcot-Marie-Tooth disease, a hereditary sensory and motor neuropathy characterized by progressive loss of the peripheral nerves. This underscores the pathophysiological importance of E4-mediated ubiquitylation in neurodegeneration. In summary, we identify E4-dependent mitochondrial stress adaptation by linking various metabolicprocesses to mitochondrial fusion and fission dynamics.

Pathogen-specific structural features of Candida albicans Ras1 activation complex: uncovering new antifungal drug targets.

An important feature associated with Candida albicans pathogenicity is its ability to switch between yeast and hyphal forms, a process in which CaRas1 plays a key role. CaRas1 is activated by the guanine nucleotide exchange factor (GEF) CaCdc25, triggering hyphal growth-related signaling pathways through its conserved GTP-binding (G)-domain. An important function in hyphal growth has also been proposed for the long hypervariable region downstream the G-domain, whose unusual content of polyglutamine stretches and Q/N repeats make CaRas1 unique within Ras proteins. Despite its biological importance, both the structure of CaRas1 and the molecular basis of its activation by CaCdc25 remain unexplored. Here, we show that CaRas1 has an elongated shape and limited conformational flexibility and that its hypervariable region contains helical structural elements, likely forming an intramolecular coiled-coil. Functional assays disclosed that CaRas1-activation by CaCdc25 is highly efficient, with activities up to 2,000-fold higher than reported for human GEFs. The crystal structure of the CaCdc25 catalytic region revealed an active conformation for the α-helical hairpin, critical for CaRas1-activation, unveiling a specific region exclusive to CTG-clade species. Structural studies on CaRas1/CaCdc25 complexes also revealed an interaction surface clearly distinct from that of homologous human complexes. Furthermore, we identified an inhibitory synthetic peptide, prompting the proposal of a key regulatory mechanism for CaCdc25. To our knowledge, this is the first report of specific inhibition of the CaRas1-activation via targeting its GEF. This, together with their unique pathogen-structural features, disclose a set of novel strategies to specifically block this important virulence-related mechanism. IMPORTANCE Candida albicans is the main causative agent of candidiasis, the commonest fungal infection in humans. The eukaryotic nature of C. albicans and the rapid emergence of antifungal resistance raise the challenge of identifying novel drug targets to battle this prevalent and life-threatening disease. CaRas1 and CaCdc25 are key players in the activation of signaling pathways triggering multiple virulence traits, including the yeast-to-hypha interconversion. The structural similarity of the conserved G-domain of CaRas1 to those of human homologs and the lack of structural information on CaCdc25 has impeded progress in targeting these proteins. The unique structural and functional features for CaRas1 and CaCdc25 presented here, together with the identification of a synthetic peptide capable of specifically inhibiting the GEF activity of CaCdc25, open new possibilities to uncover new antifungal drug targets against C. albicans virulence.

PACAP activates MRGPRX2 on meningeal mast cells to drive migraine-like pain

Migraine ranks among the most prevalent disorders worldwide, leading to disability and decreased quality of life in patients. Recently, neurogenic inflammation has been recognized as a potential underlying pathology contributing to the migraine pain pathway. Mast cells reside in the meninges and have been implicated in contributing to the pathophysiology of migraine. Here we report for the first time that the mouse Mas-Related G-protein-coupled Receptor B2 (MrgprB2), is expressed on meningeal connective tissue mast cells and contributes to Pituitary Adenylate Cyclase Activating Peptide (PACAP)-induced migraine-like pain behavior. We also found that PACAP was able to dose-dependently lead to enzyme release from human mast cells via activation of MRGPRX2; the human homolog of MrgprB2. Using a transgenic MRGPRX2 mouse, we observed significant increases in PACAP-induced migraine-like pain behavior in MRGPRX2+ mice vs mice lacking the receptor. These results reveal both MrgprB2 and MRGPRX2 as important contributors to neuropeptide-induced migraine pain.

Solution structure of the N-terminal extension domain of a Schistosoma japonicum asparaginyl-tRNA synthetase

Several secreted proteins from helminths (parasitic worms) have been shown to have immunomodulatory activities. Asparaginyl-tRNA synthetases are abundantly secreted in the filarial nematode Brugia malayi (BmAsnRS) and the parasitic flatworm Schistosoma japonicum (SjAsnRS), indicating a possible immune function. The suggestion is supported by BmAsnRS alleviating disease symptoms in a T-cell transfer mouse model of colitis. This immunomodulatory function is potentially related to an N-terminal extension domain present in eukaryotic AsnRS proteins but few structure/function studies have been done on this domain. Here we have determined the three-dimensional solution structure of the N-terminal extension domain of SjAsnRS. A protein containing the 114 N-terminal amino acids of SjAsnRS was recombinantly expressed with isotopic labelling to allow structure determination using 3D NMR spectroscopy, and analysis of dynamics using NMR relaxation experiments. Structural comparisons of the N-terminal extension domain of SjAsnRS with filarial and human homologues highlight a high degree of variability in the β-hairpin region of these eukaryotic N-AsnRS proteins, but similarities in the disorder of the C-terminal regions. Limitations in PrDOS-based intrinsically disordered region (IDR) model predictions were also evident in this comparison. Empirical structural data such as that presented in our study for N-SjAsnRS will enhance the prediction of sequence-homology based structure modelling and prediction of IDRs in the future. Communicated by Ramaswamy H. Sarma

Structure-based drug designing against Leishmania donovani using docking and molecular dynamics simulation studies: exploring glutathione synthetase as a drug target

In the pursuit of developing novel anti-leishmanial agents, we conducted an extensive computational study to screen inhibitors from the FDA-approved ZINC database against Leishmania donovani glutathione synthetase. The three-dimensional structure of Leishmania donovani glutathione synthetase was constructed by homology modeling, using the crystallographic structure of Trypanosoma brucei glutathione synthetase as a template. Subsequently, molecular docking studies were carried out for a large number of compounds using AutoDock Vina. Among the screened compounds, we selected the top five with strong binding affinity to Leishmania donovani glutathione synthetase but having a very low affinity to its human homolog. Further investigations on protein-ligand complexes were done by conducting molecular dynamics (MD) simulation and MM/PBSA analysis. The results revealed that Olysio (Simeprevir) exhibited the lowest binding energy (-89.21 kcal/mol), followed by Telithromycin (-45.34 kcal/mol). These findings showed that these compounds have the potential to act as inhibitors of glutathione synthetase. Hence, our study provides valuable insights for the development of a novel therapeutic strategy against Leishmania donovani by targeting the glutathione synthetase enzyme. Communicated by Ramaswamy H. Sarma

Identification of small molecule antivirals against HTLV-1 by targeting the hDLG1-Tax-1 protein-protein interaction

Human T-cell leukemia virus type-1 (HTLV-1) is the first pathogenic retrovirus discovered in human. Although HTLV-1-induced diseases are well-characterized and linked to the encoded Tax-1 oncoprotein, there is currently no strategy to target Tax-1 functions with small molecules. Here, we analyzed the binding of Tax-1 to the human homolog of the drosophila discs large tumor suppressor (hDLG1/SAP97), a multi-domain scaffolding protein involved in Tax-1-transformation ability. We have solved the structures of the PDZ binding motif (PBM) of Tax-1 in complex with the PDZ1 and PDZ2 domains of hDLG1 and assessed the binding of 10 million molecules by virtual screening. Among the 19 experimentally confirmed compounds, one systematically inhibited the Tax-1-hDLG1 interaction in different biophysical and cellular assays, as well as HTLV-1 cell-to-cell transmission in a T-cell model. Thus, our work demonstrates that interactions involving Tax-1 PDZ-domains are amenable to small-molecule inhibition, which provides a framework for the design of targeted therapies for HTLV-1-induced diseases.

Human nucleolar protein SURF6/RRP14 participates in early steps of pre-rRNA processing.

The biogenesis of ribosomes requires tightly controlled transcription and processing of pre-rRNA which comprises ribosomal RNAs forming the core of large and small ribosomal subunits. Early steps of the pre-rRNA processing and assembly of the ribosomal subunits require a large set of proteins that perform folding and nucleolytic cleavage of pre-rRNAs in the nucleoli. Structure and functions of proteins involved in the pre-rRNA processing have been extensively studied in the budding yeast S. cerevisiae. Functional characterization of their human homologues is complicated by the complexity of mammalian ribosomes and increased number of protein factors involved in the ribosomal biogenesis. Homologues of human nucleolar protein SURF6 from yeast and mouse, Rrp14 and Surf6, respectively, had been shown to be involved in the early steps of pre-rRNA processing. Rrp14 works as RNA chaperone in complex with proteins Ssf1 and Rrp15. Human SURF6 knockdown and overexpression were used to clarify a role of SURF6 in the early steps of pre-rRNA processing in human cell lines HeLa and HTC116. By analyzing the abundance of the rRNA precursors in cells with decreased level or overexpression of SURF6, we demonstrated that human SURF6 is involved in the maturation of rRNAs from both small and large ribosomal subunits. Changes in the SURF6 level caused by knockdown or overexpression of the protein do not result in the death of HeLa cells in contrast to murine embryonic fibroblasts, but significantly alter the distribution of cells among the phases of the cell cycle. SURF6 knockdown in both p53 sufficient and p53 deficient HCT116 human cancer cells results in elongation of G0/G1 and shortening of G2/M phase. This surprising result suggests p53 independence of SURF6 effects on the cell cycle and possible multiple functions of SURF6. Our data point to the shift from pathway 1 to pathway 2 of the rRNA biogenesis caused by the SURF6 knockdown and its likely association with p53 pathway.

Molecular dissection studies of TAC1, a transcription activator of Candida drug resistance genes of the human pathogenic fungus Candida albicans.

The up-regulation of ABC transporters Cdr1p and Cdr2p that efflux antifungal azole drugs are a leading cause of Multi-Drug Resistance (MDR) in the white fungus Candida albicans. C. albicans was reported to infect patients following the recent Covid-19 pandemic after they were given steroids for recovery. Previously, the TAC1 gene was identified as the transcriptional activator of Candida drug resistance genes (CDR1 and CDR2) and has no known human homologs. This makes it a good target for the development of novel antifungals. We, therefore, carried out the molecular dissection study of TAC1 to understand the functional regulation of the ABC transporter genes (CDR1 and CDR2) under its control. The N-terminal DNA Binding Domain (DBD) of Tac1p interacts with the Drug Responsive Element (DRE) present in the upstream promoter region of CDR1 and CDR2 genes of C. albicans. The interaction between DBD and DRE recruits Tac1p to the promoter of CDR genes. The C-terminal Acidic Activation Domain (AAD) of Tac1p interacts with the TATA box Binding Protein (TBP) and thus recruits TBP to the TATA box of CDR1 and CDR2 genes. Taking a cue from a previous study involving a TAC1 deletion strain that suggested that Tac1p acts as a xenobiotic receptor, in this study, we identified that the Middle Homology Region (MHR) of Tac1p acts as a probable xenobiotic binding domain (XBD) which plays an important role in Candida drug resistance. In addition, we studied the role of Tac1p in the regulation of some lipid profiling genes and stress response genes since they also contain the DRE consensus sequence and found that some of them can respond to xenobiotic stimuli.

SF3B1 Mutations Are Associated with Resistance to Non-Genotoxic MDM2 Inhibition in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a genetically and clinically heterogeneous malignancy affecting older individuals. There are a number of current treatment options for CLL, including monoclonal antibodies, targeted drugs, chemotherapy, and different combinations of these. However, for those patients who are intrinsically treatment resistant, or relapse following initial responses, novel targeted therapies are still needed. Targeting the mouse double-minute-2 human homolog (MDM2), a primary negative regulator of p53, is an appealing therapeutic strategy for non-genotoxic reactivation of p53, since the TP53 gene is in its wild-type state at diagnosis in approximately 90% of patients. Mutated SF3B1 and TP53 are both associated with more aggressive disease, resistance to therapies and poorer overall survival for CLL. In this study, we performed a screen for SF3B1 and TP53 mutations and tested RG7388 (idasanutlin), a second-generation MDM2 inhibitor, in a cohort of CLL primary patient samples. SF3B1 mutations were detected in 24 of 195 cases (12.3%) and found associated with poor overall survival (hazard ratio [HR] 2.12, p = 0.032) and high CD38 expression (median CD38 (%) 32 vs. 5; p = 0.0087). The novel striking finding of this study was an independent link between SF3B1 mutational status and poor response to RG7388. Overall, SF3B1 mutations in CLL patient samples were associated with resistance to treatment with RG7388 ex vivo, and patients with the wild type for both SF3B1 and TP53 are more likely to benefit from treatment with MDM2 inhibitors.

Structural basis for specific DNA sequence motif recognition by the TFAP2 transcription factors.

The TFAP2 family regulates gene expression during differentiation, development, and organogenesis, and includes five homologs in humans. They all possess a highly conserved DNA binding domain (DBD) followed by a helix-span-helix (HSH) domain. The DBD-HSH tandem domain specifically binds to a GCC(N3)GGC consensus sequence, but the precise recognition mechanisms remain unclear. Here, we found that TFAP2 preferred binding to the GCC(N3)GGC sequence, and the pseudo-palindromic GCC and GGC motifs and the length of the central spacer between the two motifs determined their binding specificity. Structural studies revealed that the two flat amphipathic α-helical HSH domains of TFAP2A stacked with each other to form a dimer via hydrophobic interactions, while the stabilized loops from both DBD domains inserted into two neighboring major grooves of the DNA duplex to form base-specific interactions. This specific DNA binding mechanism controlled the length of the central spacer and determined the DNA sequence specificity of TFAP2. Mutations of the TFAP2 proteins are implicated in various diseases. We illustrated that reduction or disruption of the DNA binding ability of the TFAP2 proteins is the primary cause of TFAP2 mutation-associated diseases. Thus, our findings also offer valuable insights into the pathogenesis of disease-associated mutations in TFAP2 proteins.

The ubiquitin-protein ligase MIEL1 localizes to peroxisomes to promote seedling oleosin degradation and lipid droplet mobilization

Lipid droplets are organelles conserved across eukaryotes that store and release neutral lipids to regulate energy homeostasis. In oilseed plants, fats stored in seed lipid droplets provide fixed carbon for seedling growth before photosynthesis begins. As fatty acids released from lipid droplet triacylglycerol are catabolized in peroxisomes, lipid droplet coat proteins are ubiquitinated, extracted, and degraded. In Arabidopsis seeds, the predominant lipid droplet coat protein is OLEOSIN1 (OLE1). To identify genes modulating lipid droplet dynamics, we mutagenized a line expressing mNeonGreen-tagged OLE1 expressed from the OLE1 promoter and isolated mutants with delayed oleosin degradation. From this screen, we identified four miel1 mutant alleles. MIEL1 (MYB30-interacting E3 ligase 1) targets specific MYB transcription factors for degradation during hormone and pathogen responses [D. Marino et al., Nat. Commun. 4, 1476 (2013); H. G. Lee and P. J. Seo, Nat. Commun. 7, 12525 (2016)] but had not been implicated in lipid droplet dynamics. OLE1 transcript levels were unchanged in miel1 mutants, indicating that MIEL1 modulates oleosin levels posttranscriptionally. When overexpressed, fluorescently tagged MIEL1 reduced oleosin levels, causing very large lipid droplets. Unexpectedly, fluorescently tagged MIEL1 localized to peroxisomes. Our data suggest that MIEL1 ubiquitinates peroxisome-proximal seed oleosins, targeting them for degradation during seedling lipid mobilization. The human MIEL1 homolog (PIRH2; p53-induced protein with a RING-H2 domain) targets p53 and other proteins for degradation and promotes tumorigenesis [A. Daks et al., Cells 11, 1515 (2022)]. When expressed in Arabidopsis, human PIRH2 also localized to peroxisomes, hinting at a previously unexplored role for PIRH2 in lipid catabolism and peroxisome biology in mammals.