Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase.

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype.

The relationship between sequence variation and phenotype is poorly understood. Here, we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in dihydrofolate reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis-Menten-like in vitro activity curve of thymidylate kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis-Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system-level implications for bacterial phenotype.

Paradoxical Role of AT-rich Interactive Domain 1A in Restraining Pancreatic Carcinogenesis.

Background & Aims: ARID1A is postulated to be a tumor suppressor gene owing to loss-of-function mutations in human pancreatic ductal adenocarcinomas (PDAC). However, its role in pancreatic pathogenesis is not clear despite recent studies using genetically engineered mouse (GEM) models. We aimed at further understanding of its direct functional role in PDAC, using a combination of GEM model and PDAC cell lines. Methods: Pancreas-specific mutant Arid1a-driven GEM model (Ptf1a-Cre; KrasG12D; Arid1af/f or "KAC") was generated by crossing Ptf1a-Cre; KrasG12D ("KC") mice with Arid1af/f mice and characterized histologically with timed necropsies. Arid1a was also deleted using CRISPR-Cas9 system in established human and murine PDAC cell lines to study the immediate effects of Arid1a loss in isogenic models. Cell lines with or without Arid1a expression were developed from respective autochthonous PDAC GEM models, compared functionally using various culture assays, and subjected to RNA-sequencing for comparative gene expression analysis. DNA damage repair was analyzed in cultured cells using immunofluorescence and COMET assay. Results: Retention of Arid1a is critical for early progression of mutant Kras-driven pre-malignant lesions into PDAC, as evident by lower Ki-67 and higher apoptosis staining in "KAC" as compared to "KC" mice. Enforced deletion of Arid1a in established PDAC cell lines caused suppression of cellular growth and migration, accompanied by compromised DNA damage repair. Despite early development of relatively indolent cystic precursor lesions called intraductal papillary mucinous neoplasms (IPMNs), a subset of "KAC" mice developed aggressive PDAC in later ages. PDAC cells obtained from older autochthonous "KAC" mice revealed various compensatory ("escaper") mechanisms to overcome the growth suppressive effects of Arid1a loss. Conclusions: Arid1a is an essential survival gene whose loss impairs cellular growth, and thus, its expression is critical during early stages of pancreatic tumorigenesis in mouse models. In tumors that arise in the setting of ARID1A loss, a multitude of "escaper" mechanisms drive progression.

Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts.

Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation.

Substrate inhibition imposes fitness penalty at high protein stability.

Proteins are only moderately stable. It has long been debated whether this narrow range of stabilities is solely a result of neutral drift toward lower stability or purifying selection against excess stability-for which no experimental evidence was found so far-is also at work. Here, we show that mutations outside the active site in the essential Escherichia coli enzyme adenylate kinase (Adk) result in a stability-dependent increase in substrate inhibition by AMP, thereby impairing overall enzyme activity at high stability. Such inhibition caused substantial fitness defects not only in the presence of excess substrate but also under physiological conditions. In the latter case, substrate inhibition caused differential accumulation of AMP in the stationary phase for the inhibition-prone mutants. Furthermore, we show that changes in flux through Adk could accurately describe the variation in fitness effects. Taken together, these data suggest that selection against substrate inhibition and hence excess stability may be an important factor determining stability observed for modern-day Adk.

Cytokine-Regulated Phosphorylation and Activation of TET2 by JAK2 in Hematopoiesis.

Even though the Ten-eleven translocation (TET) enzymes catalyze the generation of 5-hydroxymethylcytosines required for lineage commitment and subsequent differentiation of stem cells into erythroid cells, the mechanisms that link extracellular signals to TET activation and DNA hydroxymethylation are unknown. We demonstrate that hematopoietic cytokines phosphorylate TET2, leading to its activation in erythroid progenitors. Specifically, cytokine receptor-associated JAK2 phosphorylates TET2 at tyrosines 1939 and 1964. Phosphorylated TET2 interacts with the erythroid transcription factor KLF1, and this interaction with TET2 is increased upon exposure to erythropoietin. The activating JAK2V617F mutation seen in myeloproliferative disease patient samples and in mouse models is associated with increased TET activity and cytosine hydroxymethylation as well as genome-wide loss of cytosine methylation. These epigenetic and functional changes are also associated with increased expression of several oncogenic transcripts. Thus, we demonstrate that JAK2-mediated TET2 phosphorylation provides a mechanistic link between extracellular signals and epigenetic changes during hematopoiesis. SIGNIFICANCE: Identification of TET2 phosphorylation and activation by cytokine-stimulated JAK2 links extracellular signals to chromatin remodeling during hematopoietic differentiation. This provides potential avenues to regulate TET2 function in the context of myeloproliferative disorders and myelodysplastic syndromes associated with the JAK2V617F-activating mutation.This article is highlighted in the In This Issue feature, p. 681.

Ascorbic acid-induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma.

Although clear cell renal cell carcinoma (ccRCC) has been shown to result in widespread aberrant cytosine methylation and loss of 5-hydroxymethylcytosine (5hmC), the prognostic impact and therapeutic targeting of this epigenetic aberrancy has not been fully explored. Analysis of 576 primary ccRCC samples demonstrated that loss of 5hmC was strongly associated with aggressive clinicopathologic features and was an independent adverse prognostic factor. Loss of 5hmC also predicted reduced progression-free survival after resection of nonmetastatic disease. The loss of 5hmC in ccRCC was not due to mutational or transcriptional inactivation of ten eleven translocation (TET) enzymes, but to their functional inactivation by l-2-hydroxyglutarate (L2HG), which was overexpressed due to the deletion and underexpression of L2HG dehydrogenase (L2HGDH). Ascorbic acid (AA) reduced methylation and restored genome-wide 5hmC levels via TET activation. Fluorescence quenching of the recombinant TET-2 protein was unaffected by L2HG in the presence of AA. Pharmacologic AA treatment led to reduced growth of ccRCC in vitro and reduced tumor growth in vivo, with increased intratumoral 5hmC. These data demonstrate that reduced 5hmC is associated with reduced survival in ccRCC and provide a preclinical rationale for exploring the therapeutic potential of high-dose AA in ccRCC.

Antisense STAT3 inhibitor decreases viability of myelodysplastic and leukemic stem cells.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.

Design, display and immunogenicity of HIV1 gp120 fragment immunogens on virus-like particles.

The broadly neutralizing antibody against HIV-1, b12, binds to the CD4 binding site (CD4bs) on the outer domain (OD) of the gp120 subunit of HIV-1 Env. We have previously reported the design of an E. coli expressed fragment of HIV-1 gp120, b122a, containing about 70% of the b12 epitope with the idea of focusing the immune response to this structure. Since the b122a structure was found to be only partially folded, as assessed by circular dichroism and protease resistance, we attempted to stabilize it by the introduction of additional disulfide bonds. One such mutant, b122a1-b showed increased stability and bound b12 with 30-fold greater affinity as compared to b122a. Various b122a and OD fragment proteins were displayed on the surface of Qß virus-like particles. Sera raised against these particles in six-month long rabbit immunization studies could neutralize Tier1 viruses across different subtypes with the best results observed with b122a1-b displayed particles. Significantly higher amounts of antibodies directed towards the CD4bs were also elicited by particles displaying b122a1-b. This study highlights the ability of fragment immunogens to focus the antibody response to the conserved CD4bs of HIV-1.

Bacterially expressed HIV-1 gp120 outer-domain fragment immunogens with improved stability and affinity for CD4-binding site neutralizing antibodies.

Protein minimization is an attractive approach for designing vaccines against rapidly evolving pathogens such as human immunodeficiency virus, type 1 (HIV-1), because it can help in focusing the immune response toward conserved conformational epitopes present on complex targets. The outer domain (OD) of HIV-1 gp120 contains epitopes for a large number of neutralizing antibodies and therefore is a primary target for structure-based vaccine design. We have previously designed a bacterially expressed outer-domain immunogen (ODEC) that bound CD4-binding site (CD4bs) ligands with 3-12 µm affinity and elicited a modest neutralizing antibody response in rabbits. In this study, we have optimized ODEC using consensus sequence design, cyclic permutation, and structure-guided mutations to generate a number of variants with improved yields, biophysical properties, stabilities, and affinities (KD of 10-50 nm) for various CD4bs targeting broadly neutralizing antibodies, including the germline-reverted version of the broadly neutralizing antibody VRC01. In contrast to ODEC, the optimized immunogens elicited high anti-gp120 titers in rabbits as early as 6 weeks post-immunization, before any gp120 boost was given. Following two gp120 boosts, sera collected at week 22 showed cross-clade neutralization of tier 1 HIV-1 viruses. Using a number of different prime/boost combinations, we have identified a cyclically permuted OD fragment as the best priming immunogen, and a trimeric, cyclically permuted gp120 as the most suitable boosting molecule among the tested immunogens. This study also provides insights into some of the biophysical correlates of improved immunogenicity.

Accessibility of the Shine-Dalgarno Sequence Dictates N-Terminal Codon Bias in E. coli.

Despite considerable efforts, no physical mechanism has been shown to explain N-terminal codon bias in prokaryotic genomes. Using a systematic study of synonymous substitutions in two endogenous E. coli genes, we show that interactions between the coding region and the upstream Shine-Dalgarno (SD) sequence modulate the efficiency of translation initiation, affecting both intracellular mRNA and protein levels due to the inherent coupling of transcription and translation in E. coli. We further demonstrate that far-downstream mutations can also modulate mRNA levels by occluding the SD sequence through the formation of non-equilibrium secondary structures. By contrast, a non-endogenous RNA polymerase that decouples transcription and translation largely alleviates the effects of synonymous substitutions on mRNA levels. Finally, a complementary statistical analysis of the E. coli genome specifically implicates avoidance of intra-molecular base pairing with the SD sequence. Our results provide general physical insights into the coding-level features that optimize protein expression in prokaryotes.

Altered hydroxymethylation is seen at regulatory regions in pancreatic cancer and regulates oncogenic pathways.

Transcriptional deregulation of oncogenic pathways is a hallmark of cancer and can be due to epigenetic alterations. 5-Hydroxymethylcytosine (5-hmC) is an epigenetic modification that has not been studied in pancreatic cancer. Genome-wide analysis of 5-hmC-enriched loci with hmC-seal was conducted in a cohort of low-passage pancreatic cancer cell lines, primary patient-derived xenografts, and pancreatic controls and revealed strikingly altered patterns in neoplastic tissues. Differentially hydroxymethylated regions preferentially affected known regulatory regions of the genome, specifically overlapping with known H3K4me1 enhancers. Furthermore, base pair resolution analysis of cytosine methylation and hydroxymethylation with oxidative bisulfite sequencing was conducted and correlated with chromatin accessibility by ATAC-seq and gene expression by RNA-seq in pancreatic cancer and control samples. 5-hmC was specifically enriched at open regions of chromatin, and gain of 5-hmC was correlated with up-regulation of the cognate transcripts, including many oncogenic pathways implicated in pancreatic neoplasia, such as MYC, KRAS, VEGFA, and BRD4 Specifically, BRD4 was overexpressed and acquired 5-hmC at enhancer regions in the majority of neoplastic samples. Functionally, acquisition of 5-hmC at BRD4 promoter was associated with increase in transcript expression in reporter assays and primary samples. Furthermore, blockade of BRD4 inhibited pancreatic cancer growth in vivo. In summary, redistribution of 5-hmC and preferential enrichment at oncogenic enhancers is a novel regulatory mechanism in human pancreatic cancer.

Optimization of lag phase shapes the evolution of a bacterial enzyme.

Mutations provide the variation that drives evolution, yet their effects on fitness remain poorly understood. Here we explore how mutations in the essential enzyme adenylate kinase (Adk) of Escherichia coli affect multiple phases of population growth. We introduce a biophysical fitness landscape for these phases, showing how they depend on molecular and cellular properties of Adk. We find that Adk catalytic capacity in the cell (the product of activity and abundance) is the major determinant of mutational fitness effects. We show that bacterial lag times are at a well-defined optimum with respect to Adk's catalytic capacity, while exponential growth rates are only weakly affected by variation in Adk. Direct pairwise competitions between strains show how environmental conditions modulate the outcome of a competition where growth rates and lag times have a tradeoff, shedding light on the multidimensional nature of fitness and its importance in the evolutionary optimization of enzymes.

Transient protein-protein interactions perturb E. coli metabolome and cause gene dosage toxicity.

Gene dosage toxicity (GDT) is an important factor that determines optimal levels of protein abundances, yet its molecular underpinnings remain unknown. Here, we demonstrate that overexpression of DHFR in E. coli causes a toxic metabolic imbalance triggered by interactions with several functionally related enzymes. Though deleterious in the overexpression regime, surprisingly, these interactions are beneficial at physiological concentrations, implying their functional significance in vivo. Moreover, we found that overexpression of orthologous DHFR proteins had minimal effect on all levels of cellular organization - molecular, systems, and phenotypic, in sharp contrast to E. coli DHFR. Dramatic difference of GDT between 'E. coli's self' and 'foreign' proteins suggests the crucial role of evolutionary selection in shaping protein-protein interaction (PPI) networks at the whole proteome level. This study shows how protein overexpression perturbs a dynamic metabolon of weak yet potentially functional PPI, with consequences for the metabolic state of cells and their fitness.

Pexmetinib: A Novel Dual Inhibitor of Tie2 and p38 MAPK with Efficacy in Preclinical Models of Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) suppress normal hematopoietic activity in part by enabling a pathogenic inflammatory milieu in the bone marrow. In this report, we show that elevation of angiopoietin-1 in myelodysplastic CD34(+) stem-like cells is associated with higher risk disease and reduced overall survival in MDS and AML patients. Increased angiopoietin-1 expression was associated with a transcriptomic signature similar to known MDS/AML stem-like cell profiles. In seeking a small-molecule inhibitor of this pathway, we discovered and validated pexmetinib (ARRY-614), an inhibitor of the angiopoietin-1 receptor Tie-2, which was also found to inhibit the proinflammatory kinase p38 MAPK (which is overactivated in MDS). Pexmetinib inhibited leukemic proliferation, prevented activation of downstream effector kinases, and abrogated the effects of TNFα on healthy hematopoietic stem cells. Notably, treatment of primary MDS specimens with this compound stimulated hematopoiesis. Our results provide preclinical proof of concept for pexmetinib as a Tie-2/p38 MAPK dual inhibitor applicable to the treatment of MDS/AML. Cancer Res; 76(16); 4841-9. ©2016 AACR.

Protein Homeostasis Imposes a Barrier on Functional Integration of Horizontally Transferred Genes in Bacteria.

Horizontal gene transfer (HGT) plays a central role in bacterial evolution, yet the molecular and cellular constraints on functional integration of the foreign genes are poorly understood. Here we performed inter-species replacement of the chromosomal folA gene, encoding an essential metabolic enzyme dihydrofolate reductase (DHFR), with orthologs from 35 other mesophilic bacteria. The orthologous inter-species replacements caused a marked drop (in the range 10-90%) in bacterial growth rate despite the fact that most orthologous DHFRs are as stable as E.coli DHFR at 37°C and are more catalytically active than E. coli DHFR. Although phylogenetic distance between E. coli and orthologous DHFRs as well as their individual molecular properties correlate poorly with growth rates, the product of the intracellular DHFR abundance and catalytic activity (kcat/KM), correlates strongly with growth rates, indicating that the drop in DHFR abundance constitutes the major fitness barrier to HGT. Serial propagation of the orthologous strains for ~600 generations dramatically improved growth rates by largely alleviating the fitness barriers. Whole genome sequencing and global proteome quantification revealed that the evolved strains with the largest fitness improvements have accumulated mutations that inactivated the ATP-dependent Lon protease, causing an increase in the intracellular DHFR abundance. In one case DHFR abundance increased further due to mutations accumulated in folA promoter, but only after the lon inactivating mutations were fixed in the population. Thus, by apparently distinguishing between self and non-self proteins, protein homeostasis imposes an immediate and global barrier to the functional integration of foreign genes by decreasing the intracellular abundance of their products. Once this barrier is alleviated, more fine-tuned evolution occurs to adjust the function/expression of the transferred proteins to the constraints imposed by the intracellular environment of the host organism.

TET1 is a tumor suppressor of hematopoietic malignancy.

The methylcytosine dioxygenase TET1 ('ten-eleven translocation 1') is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.

Systems-level response to point mutations in a core metabolic enzyme modulates genotype-phenotype relationship.

Linking the molecular effects of mutations to fitness is central to understanding evolutionary dynamics. Here, we establish a quantitative relation between the global effect of mutations on the E. coli proteome and bacterial fitness. We created E. coli strains with specific destabilizing mutations in the chromosomal folA gene encoding dihydrofolate reductase (DHFR) and quantified the ensuing changes in the abundances of 2,000+ E. coli proteins in mutant strains using tandem mass tags with subsequent LC-MS/MS. mRNA abundances in the same E. coli strains were also quantified. The proteomic effects of mutations in DHFR are quantitatively linked to phenotype: the SDs of the distributions of logarithms of relative (to WT) protein abundances anticorrelate with bacterial growth rates. Proteomes hierarchically cluster first by media conditions, and within each condition, by the severity of the perturbation to DHFR function. These results highlight the importance of a systems-level layer in the genotype-phenotype relationship.

IL8-CXCR2 pathway inhibition as a therapeutic strategy against MDS and AML stem cells.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Novel therapeutic targets against preleukemic stem cells need to be identified for potentially curative strategies. We conducted parallel transcriptional analysis of highly fractionated stem and progenitor populations in MDS, AML, and control samples and found interleukin 8 (IL8) to be consistently overexpressed in patient samples. The receptor for IL8, CXCR2, was also significantly increased in MDS CD34(+) cells from a large clinical cohort and was predictive of increased transfusion dependence. High CXCR2 expression was also an adverse prognostic factor in The Cancer Genome Atlas AML cohort, further pointing to the critical role of the IL8-CXCR2 axis in AML/MDS. Functionally, CXCR2 inhibition by knockdown and pharmacologic approaches led to a significant reduction in proliferation in several leukemic cell lines and primary MDS/AML samples via induction of G0/G1 cell cycle arrest. Importantly, inhibition of CXCR2 selectively inhibited immature hematopoietic stem cells from MDS/AML samples without an effect on healthy controls. CXCR2 knockdown also impaired leukemic growth in vivo. Together, these studies demonstrate that the IL8 receptor CXCR2 is an adverse prognostic factor in MDS/AML and is a potential therapeutic target against immature leukemic stem cell-enriched cell fractions in MDS and AML.