Exploration of Redox-Based Functional Switching and Intermediate Substrate Channeling in Proline Catabolism

The bioaccumulation and catabolism of the amino acid proline have been shown to participate in numerous cellular processes. There is a wealth of emerging evidence that proline metabolism plays vital roles in a number of different pathogenic organisms; ranging from energy production to stress protection and beyond, as reviewed in Chapter 1. In eukaryotes and gram-positive bacteria, the catabolism of proline to form glutamate is catalyzed by two separate enzymes, proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH). In gram-negative bacteria, these two enzymes are fused into a single, multifunctional protein known as proline utilization A, or PutA. The formation of glutamate from proline is enzymatically intriguing due to the phenomena of redox-based functional switching and substrate channeling. Some PutA proteins exhibit a third functionality in addition to the typical PRODH and P5CDH activities. These trifunctional PutAs oscillate between functioning as a self-regulating transcriptional repressor and a membrane-bound enzyme, depending on the redox state of the flavin cofactor of the PRODH active site. Chapter 2 examines the role of the c-terminus of Escherichia coli PutA (EcPutA) in membrane binding, and establishes the region of PutA-membrane interactions. Chapter 3 furthers the knowledge of the mechanism of functional switching, highlighting key residues vital for the transmission of the redox-based signal from the flavin cofactor to the membrane binding domain and discusses the development of assays to examine signal transmission in vivo. Substrate channeling occurs when an intermediate substrate is transferred between two enzymes or two active sites, without equilibrating into the surrounding environment. Previous evidence demonstrates that this phenomenon occurs via a hollow cavity in PutA proteins and also occurs between monofunctional PRODH and P5CDH in vitro. Chapter 4 of this dissertation examines P5C channeling in mono- and multifunctional systems in vivo. Furthermore, substrate channeling was disrupted both between monofunctional PRODH and P5CDH from Thermus thermophilus and in PutA proteins through site-directed mutagenesis. Collectively, the research presented in this dissertation has made significant advancements in the knowledge of functional switching and substrate channeling in monofunctional and multifunctional enzymes of proline metabolism

Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A

<i>Escherichia coli</i> proline utilization A (<i>Ec</i>PutA) is the archetype of trifunctional PutA flavoproteins, which function both as regulators of the proline utilization operon and bifunctional enzymes that catalyze the four-electron oxidation of proline to glutamate. <i>Ec</i>PutA shifts from a self-regulating transcriptional repressor to a bifunctional enzyme in a process known as functional switching. The flavin redox state dictates the function of <i>Ec</i>PutA. Upon proline oxidation, the flavin becomes reduced, triggering a conformational change that causes <i>Ec</i>PutA to dissociate from the <i>put</i> regulon and bind to the cellular membrane. Major structure/function domains of <i>Ec</i>PutA have been characterized, including the DNA-binding domain, proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase catalytic domains, and an aldehyde dehydrogenase superfamily fold domain. Still lacking is an understanding of the membrane-binding domain, which is essential for <i>Ec</i>PutA catalytic turnover and functional switching. Here, we provide evidence for a conserved C-terminal motif (CCM) in <i>Ec</i>PutA having a critical role in membrane binding. Deletion of the CCM or replacement of hydrophobic residues with negatively charged residues within the CCM impairs <i>Ec</i>PutA functional and physical membrane association. Furthermore, cell-based transcription assays and limited proteolysis indicate that the CCM is essential for functional switching. Using fluorescence resonance energy transfer involving dansyl-labeled liposomes, residues in the α-domain are also implicated in membrane binding. Taken together, these experiments suggest that the CCM and α-domain converge to form a membrane-binding interface near the PRODH domain. The discovery of the membrane-binding region will assist efforts to define flavin redox signaling pathways responsible for <i>Ec</i>PutA functional switching

ADAR1 restricts ZBP1-mediated immune response and PANoptosis to promote tumorigenesis

Cell death provides host defense and maintains homeostasis. Za-containing molecules are essential for these processes. Z-DNA binding protein 1 (ZBP1) activates inflammatory cell death, PANoptosis, whereas adenosine deaminase acting on RNA 1 (ADAR1) serves as an RNA editor to maintain homeostasis. Here, we identify and characterize ADAR1&apos;s interaction with ZBP1, defining its role in cell death regulation and tumorigenesis. Combining interferons (IFNs) and nuclear export inhibitors (NEIs) activates ZBP1-dependent PANoptosis. ADAR1 suppresses this PANoptosis by interacting with the Z alpha 2 domain of ZBP1 to limit ZBP1 and RIPK3 interactions. Adar1(fl/fl)LysM(cre) mice are resistant to development of colorectal cancer andmelanoma, but deletion of the ZBP1 Za2 domain restores tumorigenesis in these mice. In addition, treating wild-type mice with IFN-gamma and the NEI KPT-330 regresses melanoma in a ZBP1-dependent manner. Our findings suggest that ADAR1 suppresses ZBP1-mediated PANoptosis, promoting tumorigenesis. Defining the functions of ADAR1 and ZBP1 in cell death is fundamental to informing therapeutic strategies for cancer and other diseases

Galactosaminogalactan activates the inflammasome to provide host protection

International audienceGalactosaminogalactan of Aspergillus fumigatus acts as a pathogen-associated molecular pattern that activates the NLRP3 inflammasome, which is crucial for anti-fungal host defence.Inflammasomes are important sentinels of innate immune defence that are activated in response to diverse stimuli, including pathogen-associated molecular patterns (PAMPs)(1). Activation of the inflammasome provides host defence against aspergillosis(2,3), which is a major health concern for patients who are immunocompromised. However, the Aspergillus fumigatus PAMPs that are responsible for inflammasome activation are not known. Here we show that the polysaccharide galactosaminogalactan (GAG) of A. fumigatus is a PAMP that activates the NLRP3 inflammasome. The binding of GAG to ribosomal proteins inhibited cellular translation machinery, and thus activated the NLRP3 inflammasome. The galactosamine moiety bound to ribosomal proteins and blocked cellular translation, which triggered activation of the NLRP3 inflammasome. In mice, a GAG-deficient Aspergillus mutant (Delta gt4c) did not elicit protective activation of the inflammasome, and this strain exhibited enhanced virulence. Moreover, administration of GAG protected mice from colitis induced by dextran sulfate sodium in an inflammasome-dependent manner. Thus, ribosomes connect the sensing of this fungal PAMP to the activation of an innate immune response

Evidence That the C‑Terminal Domain of a Type B PutA Protein Contributes to Aldehyde Dehydrogenase Activity and Substrate Channeling

Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ1- pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress curve for the coupled PRODH−P5CDH activity of RcPutA does not exhibit a time lag, implying a substrate channeling mechanism. RcPutA is monomeric in solution, which is unprecedented for PutAs. SAXS rigid body modeling with target−decoy validation is used to build a model of RcPutA. On the basis of homology to aldehyde dehydrogenases (ALDHs), the CTDUF is predicted to consist of a β-hairpin fused to a noncatalytic Rossmann fold domain. The predicted tertiary structural interactions of the CTDUF resemble the quaternary structural interactions in the type A PutA dimer interface. The model is tested by mutagenesis of the dimerization hairpin of a type A PutA and the CTDUF hairpin of RcPutA. Similar functional phenotypes are observed in the two sets of variants, supporting the hypothesis that the CTDUF mimics the type A PutA dimer interface. These results suggest annotation of the CTDUF as an ALDH superfamily domain that facilitates P5CDH activity and substrate channeling by stabilizing the aldehyde-binding site and sealing the substrate-channeling tunnel from the bulk medium

\u3ci\u3eDrosophila\u3c/i\u3e Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu