Sook Lee

Structural Basis of Metallo-β-Lactamase Inhibition by Captopril Stereoisomers

ABSTRACT β-Lactams are the most successful antibacterials, but their effectiveness is threatened by resistance, most importantly by production of serine- and metallo-β-lactamases (MBLs). MBLs are of increasing concern because they catalyze the hydrolysis of almost all β-lactam antibiotics, including recent-generation carbapenems. Clinically useful serine-β-lactamase inhibitors have been developed, but such inhibitors are not available for MBLs. l-Captopril, which is used to treat hypertension via angiotensin-converting enzyme inhibition, has been reported to inhibit MBLs by chelating the active site zinc ions via its thiol(ate). We report systematic studies on B1 MBL inhibition by all four captopril stereoisomers. High-resolution crystal structures of three MBLs (IMP-1, BcII, and VIM-2) in complex with either the l- or d-captopril stereoisomer reveal correlations between the binding mode and inhibition potency. The results will be useful in the design of MBL inhibitors with the breadth of selectivity required for clinical application against carbapenem-resistant Enterobacteriaceae and other organisms causing MBL-mediated resistant infections.

CAAT/enhancer-binding protein δ and cAMP-response element-binding protein mediate inducible expression of the nerve growth factor gene in the central nervous system

Nerve growth factor (NGF) synthesis in the rat cerebral cortex is induced by the β2-adrenergic receptor agonist clenbuterol (CLE). Because NGF is a crucial neurotrophic factor for basal forebrain cholinergic neurons, defining the mechanisms that regulate its transcription is important for developing therapeutic strategies to treat pathologies of these neurons. We previously showed that the transcription factor CCAAT/enhancer-binding protein δ (C/EBPδ) contributes to NGF gene regulation. Here we have further defined the function of C/EBPδ and identified a role for cAMP response element-binding protein (CREB) in NGF transcription. Inhibition of protein kinase A in C6-2B glioma cells suppressed CLE induction of an NGF promoter-reporter construct, whereas overexpression of protein kinase A increased NGF promoter activity, particularly in combination with C/EBPδ. A CRE-like site that binds CREB was identified in the proximal NGF promoter, and C/EBPδ and CREB were found to associate with the NGF promoter in vivo. Deletion of the CRE and/or C/EBP sites reduced CLE responsiveness of the promoter. In addition, ectopic expression of C/EBPδ in combination with CLE treatment increased endogenous NGF mRNA levels in C6-2B cells. C/EBPδ null mice showed complete loss of NGF induction in the cerebral cortex following CLE treatment, demonstrating a critical role for C/EBPδ in regulating β2-adrenergic receptor-mediated NGF expression in vivo. Thus, our findings demonstrate a critical role for C/EBPδ in regional expression of NGF in the brain and implicate CREB in CLE-induced NGF gene transcription.

C/EBPγ Suppresses Senescence and Inflammatory Gene Expression by Heterodimerizing with C/EBPβ

ABSTRACT C/EBPβ is an important regulator of oncogene-induced senescence (OIS). Here, we show that C/EBPγ, a heterodimeric partner of C/EBPβ whose biological functions are not well understood, inhibits cellular senescence. Cebpg−/− mouse embryonic fibroblasts (MEFs) proliferated poorly, entered senescence prematurely, and expressed a proinflammatory gene signature, including elevated levels of senescence-associated secretory phenotype (SASP) genes whose induction by oncogenic stress requires C/EBPβ. The senescence-suppressing activity of C/EBPγ required its ability to heterodimerize with C/EBPβ. Covalently linked C/EBPβ homodimers (β∼β) inhibited the proliferation and tumorigenicity of RasV12-transformed NIH 3T3 cells, activated SASP gene expression, and recruited the CBP coactivator in a Ras-dependent manner, whereas γ∼β heterodimers lacked these capabilities and efficiently rescued proliferation of Cebpg−/− MEFs. C/EBPβ depletion partially restored growth of C/EBPγ-deficient cells, indicating that the increased levels of C/EBPβ homodimers in Cebpg−/− MEFs inhibit proliferation. The proliferative functions of C/EBPγ are not restricted to fibroblasts, as hematopoietic progenitors from Cebpg−/− bone marrow also displayed impaired growth. Furthermore, high CEBPG expression correlated with poorer clinical prognoses in several human cancers, and C/EBPγ depletion decreased proliferation and induced senescence in lung tumor cells. Our findings demonstrate that C/EBPγ neutralizes the cytostatic activity of C/EBPβ through heterodimerization, which prevents senescence and suppresses basal transcription of SASP genes.

CCAAT/Enhancer-binding Protein β DNA Binding Is Auto-inhibited by Multiple Elements That Also Mediate Association with p300/CREB-binding Protein (CBP)

Signaling through Ras GTPases controls the activity of many transcription factors including CCAAT/enhancer-binding protein (C/EBPβ), which regulates oncogenic H-RasV12-induced senescence and growth arrest. Here we report that C/EBPβ (LAP) DNA binding is inhibited by N-terminal sequences and derepressed by oncogenic Ras signaling. Sequence and mutational analyses showed that auto-repression involves two LXXLF (φXXφφ)-like motifs (LX1 and LX2) and a third element, auto-inhibitory domain (AID), located within conserved region CR5. LX1 is a critical component of the transactivation domain and has been shown to mediate C/EBPβ binding to the TAZ2 region of p300/CREB-binding protein coactivators. C/EBPβ auto-repression also involves a C-terminal regulatory domain (CRD) adjacent to the leucine zipper. CRD contains a third φXXφφ motif (LX3) and a short sequence, KQL, which has similarity to a region in the protein-binding site of TAZ2. The C/EBPβ N- and C-terminal domains physically associate in a manner that requires the basic region and CRD. We propose a model in which the regulatory sequences form a hydrophobic core that reciprocally inhibits DNA binding and transactivation. We also suggest a mechanism for C/EBPβ derepression involving several recently identified modifications within AID and CRD. Finally, we show that association of activated C/EBPβ with p300/CREB-binding protein requires the LX2 and AID auto-inhibitory elements. Thus, the N-terminal regulatory elements have dual roles in auto-inhibition and coactivator binding.

3′UTR elements inhibit Ras-induced C/EBPβ post-translational activation and senescence in tumour cells

C/EBPβ is an auto‐repressed protein that becomes post‐translationally activated by Ras‐MEK‐ERK signalling. C/EBPβ is required for oncogene‐induced senescence (OIS) of primary fibroblasts, but also displays pro‐oncogenic functions in many tumour cells. Here, we show that C/EBPβ activation by H‐RasV12 is suppressed in immortalized/transformed cells, but not in primary cells, by its 3′ untranslated region (3′UTR). 3′UTR sequences inhibited Ras‐induced cytostatic activity of C/EBPβ, DNA binding, transactivation, phosphorylation, and homodimerization, without significantly affecting protein expression. The 3′UTR suppressed induction of senescence‐associated C/EBPβ target genes, while promoting expression of genes linked to cancers and TGFβ signalling. An AU‐rich element (ARE) and its cognate RNA‐binding protein, HuR, were required for 3′UTR inhibition. These components also excluded the Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPβ translation controls de‐repression by Ras signalling. Notably, 3′UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras‐induced C/EBPβ activation and OIS to proceed. Our findings reveal a novel mechanism whereby non‐coding mRNA sequences selectively regulate C/EBPβ activity and suppress its anti‐oncogenic functions.

CCAAT/enhancer binding protein β DNA binding is auto-inhibited by multiple elements that also mediate association with p300/CBP

Signaling through Ras GTPases controls the activity of many transcription factors including CCAAT/enhancer-binding protein (C/EBPβ), which regulates oncogenic H-RasV12-induced senescence and growth arrest. Here we report that C/EBPβ (LAP) DNA binding is inhibited by N-terminal sequences and derepressed by oncogenic Ras signaling. Sequence and mutational analyses showed that auto-repression involves two LXXLF (φXXφφ)-like motifs (LX1 and LX2) and a third element, auto-inhibitory domain (AID), located within conserved region CR5. LX1 is a critical component of the transactivation domain and has been shown to mediate C/EBPβ binding to the TAZ2 region of p300/CREB-binding protein coactivators. C/EBPβ auto-repression also involves a C-terminal regulatory domain (CRD) adjacent to the leucine zipper. CRD contains a third φXXφφ motif (LX3) and a short sequence, KQL, which has similarity to a region in the protein-binding site of TAZ2. The C/EBPβ N- and C-terminal domains physically associate in a manner that requires the basic region and CRD. We propose a model in which the regulatory sequences form a hydrophobic core that reciprocally inhibits DNA binding and transactivation. We also suggest a mechanism for C/EBPβ derepression involving several recently identified modifications within AID and CRD. Finally, we show that association of activated C/EBPβ with p300/CREB-binding protein requires the LX2 and AID auto-inhibitory elements. Thus, the N-terminal regulatory elements have dual roles in auto-inhibition and coactivator binding.

The Chemical Biology of Human Metallo-β-Lactamase Fold Proteins.

The αββα metallo β-lactamase (MBL) fold (MBLf) was first observed in bacterial enzymes that catalyze the hydrolysis of almost all β-lactam antibiotics, but is now known to be widely distributed. The MBL core protein fold is present in human enzymes with diverse biological roles, including cell detoxification pathways and enabling resistance to clinically important anticancer medicines. Human (h)MBLf enzymes can bind metals, including zinc and iron ions, and catalyze a range of chemically interesting reactions, including both redox (e.g., ETHE1) and hydrolytic processes (e.g., Glyoxalase II, SNM1 nucleases, and CPSF73). With a view to promoting basic research on MBLf enzymes and their medicinal targeting, here we summarize current knowledge of the mechanisms and roles of these important molecules.

Characterization of the Human SNM1A and SNM1B/Apollo DNA Repair Exonucleases

Background: The nucleases hSNM1A and hSNM1B are implicated in DNA interstrand cross-link repair. Results: hSNM1A and hSNM1B were biochemically characterized using undamaged and cross-linked DNA. A real-time assay for the nucleases suitable for inhibitor identification was developed. Conclusion: Preferential hSNM1A activation by high molecular weight and cross-linked DNA was observed. Significance: This work provides a basis for hSNM1A inhibitor development for improved cancer therapy. Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5′-to-3′ directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.

CSB interacts with SNM1A and promotes DNA interstrand crosslink processing

Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5′-3′ exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins—with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro—and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.

The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.