Functional interactions between NADPH oxidase 5 and actin

NADPH oxidase 5 (NOX5) is a transmembrane oxidative signaling enzyme which produces superoxide in response to intracellular calcium flux. Increasing evidence indicates that NOX5 is involved in a variety of physiological processes as well as human disease, however, details of NOX5 signaling pathways and targets of NOX5 mediated oxidative modifications remain poorly resolved. Actin dynamics have previously been shown to be modulated by oxidative modification, however, a direct connection to NOX5 expression and activity has not been fully explored. Here we show that NOX5 and actin interact in the cell, and each modulate the activity of the other. Using actin effector molecules jasplakinolide, cytochalasin D and latrunculin A, we show that changes in actin dynamics affect NOX5 superoxide production. In tandem, NOX5 oxidatively modifies actin, and shifts the ratio of filamentous to monomeric actin. Finally, we show that knockdown of NOX5 in the pancreatic cancer cell line PSN-1 impairs cell migration. Together our findings indicate an important link between actin dynamics and oxidative signaling through NOX5

Mining Disaggregase Sequence Space to Safely Counter TDP-43, FUS, and α-Synuclein Proteotoxicity.

Hsp104 is an AAA+ protein disaggregase, which can be potentiated via diverse mutations in its autoregulatory middle domain (MD) to mitigate toxic misfolding of TDP-43, FUS, and α-synuclein implicated in fatal neurodegenerative disorders. Problematically, potentiated MD variants can exhibit off-target toxicity. Here, we mine disaggregase sequence space to safely enhance Hsp104 activity via single mutations in nucleotide-binding domain 1 (NBD1) or NBD2. Like MD variants, NBD variants counter TDP-43, FUS, and α-synuclein toxicity and exhibit elevated ATPase and disaggregase activity. Unlike MD variants, non-toxic NBD1 and NBD2 variants emerge that rescue TDP-43, FUS, and α-synuclein toxicity. Potentiating substitutions alter NBD1 residues that contact ATP, ATP-binding residues, or the MD. Mutating the NBD2 protomer interface can also safely ameliorate Hsp104. Thus, we disambiguate allosteric regulation of Hsp104 by several tunable structural contacts, which can be engineered to spawn enhanced therapeutic disaggregases with minimal off-target toxicity

Adolescence and the next generation.

Adolescent growth and social development shape the early development of offspring from preconception through to the post-partum period through distinct processes in males and females. At a time of great change in the forces shaping adolescence, including the timing of parenthood, investments in today's adolescents, the largest cohort in human history, will yield great dividends for future generations

Structural and mechanistic insights into the yeast disaggregase Hsp104

Hsp104 is a hexameric, AAA+ disaggregase from yeast, which couples ATP hydrolysis to remodeling diverse substrates ranging from amorphous aggregates to amyloid fibers. A mechanistic understanding of Hsp104\u27s substrate remodeling activities remains poorly defined. The hexamer undergoes large conformational changes upon ATP hydrolysis, but the details of these changes and how they are coupled to substrate remodeling are unresolved. The goals of this thesis were to elucidate low and high-resolution structural information about the Hsp104 hexamer and to discover new details of the mechanism of substrate remodeling. We used the in solution structural techniques small angle x-ray scattering and synchrotron x-ray footprinting, complemented by several biochemical assays, to elucidate novel roles for several Hsp104 domains, and to develop a comprehensive model for how the Hsp104 hexamer engages substrate and unleashes its remodeling capabilities. We discovered that the N-terminal domain (NTD) is involved in substrate binding, productive interactions with Hsp70, and an interface with nucleotide binding domain 1 (NBD1) and the middle domain (MD). We discovered a loop in NBD1 that may engage substrate in the ADP bound state to prevent premature substrate release, identified the region of the MD (helix 2) responsible and the mechanism of signal transmission between NBD1 and NBD2, and confirmed the validity of a hexameric model of the NBD2 domain. Hsp104 engages substrate in the ATP-bound state. We have found that in this state Hsp104 displays an increase in rigidity, which we propose as a pre-payment of the entropic cost of substrate binding. Initial substrate engagement in the NTD and NBD1, as well as Hsp70 interactions at the NTD:NBD1:MD interface, serve to `prime the pump\u27. These interactions result in large conformational changes of the MD, specifically in helix 2, which spans the entirety of the domain. These conformational changes increase MD dynamics, partially releasing MD:NBD2 contacts, and allow signal transmission between NBD1 and NBD2. As NBD2 responds to these signals, a positive feedback loop is created. Further nucleotide binding in NBD2 stimulates ATP hydrolysis in NBD1, and substrate is remodeled by iterative binding events and peristaltic motions of the Hsp104 hexamer channel

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of <i>Saccharomyces cerevisiae</i>

Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N₂ fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O₂ consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of <i>Saccharomyces cerevisiae</i> in which transcriptional units comprising nine <i>Azotobacter vinelandii nif</i> genes (<i>nifHDKUSMBEN</i>) were integrated into the genome. Two combinatorial libraries of <i>nif</i> gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of <i>nif</i> gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and <i>nif</i> expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust <i>nif</i> gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell

Mining Disaggregase Sequence Space to Safely Counter TDP-43, FUS, and α-Synuclein Proteotoxicity.

Hsp104 is an AAA+ protein disaggregase, which can be potentiated via diverse mutations in its autoregulatory middle domain (MD) to mitigate toxic misfolding of TDP-43, FUS, and α-synuclein implicated in fatal neurodegenerative disorders. Problematically, potentiated MD variants can exhibit off-target toxicity. Here, we mine disaggregase sequence space to safely enhance Hsp104 activity via single mutations in nucleotide-binding domain 1 (NBD1) or NBD2. Like MD variants, NBD variants counter TDP-43, FUS, and α-synuclein toxicity and exhibit elevated ATPase and disaggregase activity. Unlike MD variants, non-toxic NBD1 and NBD2 variants emerge that rescue TDP-43, FUS, and α-synuclein toxicity. Potentiating substitutions alter NBD1 residues that contact ATP, ATP-binding residues, or the MD. Mutating the NBD2 protomer interface can also safely ameliorate Hsp104. Thus, we disambiguate allosteric regulation of Hsp104 by several tunable structural contacts, which can be engineered to spawn enhanced therapeutic disaggregases with minimal off-target toxicity