Time to split: biomarker trajectories in pediatric acute respiratory distress syndrome hint at underlying disease.

Pediatric acute respiratory distress syndrome (ARDS) is severe, noncardiac hypoxemic respiratory failure that carries a substantial risk of death. Given the complexity of this clinically defined syndrome and the repeated failure of therapeutic trials, there has been an effort to identify subphenotypes of ARDS that may share targetable mechanisms of disease. In this issue of the JCI, Yehya and colleagues measured 19 plasma biomarkers in 279 children over the first seven days of ARDS. Increases in select tissue injury makers and inflammatory cytokines in peripheral blood were associated with multiple organ dysfunction syndrome and death, but not persistent ARDS. These findings argue that splitting patients by clinical and molecular phenotype may be more informative than lumping them under the umbrella diagnosis of ARDS. However, future studies are needed to determine whether these plasma factors represent targetable pathways in lung injury or are a consequence of systemic organ dysfunction.

Comparative Analysis of Mutant Huntingtin Binding Partners in Yeast Species.

Huntington's disease is caused by the pathological expansion of a polyglutamine (polyQ) stretch in Huntingtin (Htt), but the molecular mechanisms by which polyQ expansion in Htt causes toxicity in selective neuronal populations remain poorly understood. Interestingly, heterologous expression of expanded polyQ Htt is toxic in Saccharomyces cerevisiae cells, but has no effect in Schizosaccharomyces pombe, a related yeast species possessing very few endogenous polyQ or Q/N-rich proteins. Here, we used a comprehensive and unbiased mass spectrometric approach to identify proteins that bind Htt in a length-dependent manner in both species. Analysis of the expanded polyQ-associated proteins reveals marked enrichment of proteins that are localized to and play functional roles in nucleoli and mitochondria in S. cerevisiae, but not in S. pombe. Moreover, expanded polyQ Htt appears to interact preferentially with endogenous polyQ and Q/N-rich proteins, which are rare in S. pombe, as well as proteins containing coiled-coil motifs in S. cerevisiae. Taken together, these results suggest that polyQ expansion of Htt may cause cellular toxicity in S. cerevisiae by sequestering endogenous polyQ and Q/N-rich proteins, particularly within nucleoli and mitochondria.

Nuclear Excluded Autism-Associated Phosphatase and Tensin Homolog Mutations Dysregulate Neuronal Growth.

BACKGROUND: Phosphatase and tensin homolog (PTEN) negatively regulates downstream protein kinase B signaling, resulting in decreased cellular growth and proliferation. PTEN is mutated in a subset of children with autism spectrum disorder (ASD); however, the mechanism by which specific point mutations alter PTEN function is largely unknown. Here, we assessed how ASD-associated single-nucleotide variations in PTEN (ASD-PTEN) affect function. METHODS: We used viral-mediated molecular substitution of human PTEN into Pten knockout mouse neurons and assessed neuronal morphology to determine the functional impact of ASD-PTEN. We employed molecular cloning to examine how PTEN's stability, subcellular localization, and catalytic activity affect neuronal growth. RESULTS: We identified a set of ASD-PTEN mutations displaying altered lipid phosphatase function and subcellular localization. We demonstrated that wild-type PTEN can rescue the neuronal hypertrophy, while PTEN H93R, F241S, D252G, W274L, N276S, and D326N failed to rescue this hypertrophy. A subset of these mutations lacked nuclear localization, prompting us to examine the role of nuclear PTEN in regulating neuronal growth. We found that nuclear PTEN alone is sufficient to regulate soma size. Furthermore, forced localization of the D252G and W274L mutations into the nucleus partially restores regulation of soma size. CONCLUSIONS: ASD-PTEN mutations display decreased stability, catalytic activity, and/or altered subcellular localization. Mutations lacking nuclear localization uncover a novel mechanism whereby lipid phosphatase activity in the nucleus can regulate mammalian target of rapamycin signaling and neuronal growth.

CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins.

Proteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt) with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington's disease (HD). To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt) in Schizosaccharomyces pombe In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms. IMPORTANCE: Polyglutamine (polyQ) proteins encoded by repetitive CAG DNA sequences serve a variety of normal biological functions. Yet some proteins with abnormally expanded polyQ regions cause neurodegeneration through unknown mechanisms. To study how distinct cellular environments modulate polyQ aggregation and toxicity, we expressed CAG-expanded huntingtin fragments in Schizosaccharomyces pombe In stark contrast to many other eukaryotes, S. pombe is uniquely devoid of proteins containing long polyQ tracts. Our results show that S. pombe cells, despite their low content of endogenous polyQ proteins, exhibit striking and unexpected resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the emergence and expansion of polyQ domains in eukaryotic evolution.

Prion nucleation site unmasked by transient interaction with phospholipid cofactor.

Infectious mammalian prions can be formed de novo from purified recombinant prion protein (PrP) substrate through a pathway that requires the sequential addition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and RNA cofactor molecules. Recent studies show that the initial interaction between PrP and POPG causes widespread and persistent conformational changes to form an insoluble intermediate species, termed PrP(Int1). Here, we characterize the mechanism and functional consequences of the interaction between POPG and PrP. Negative-stain electron microscopy of PrP(Int1) revealed the presence of amorphous aggregates. Pull-down and photoaffinity label experiments indicate that POPG induces the formation of a PrP(C) polybasic-domain-binding neoepitope within PrP(Int1). The ongoing presence of POPG is not required to maintain PrP(Int1) structure, as indicated by the absence of stoichiometric levels of POPG in solid-state NMR measurements of PrP(Int1). Together, these results show that a transient interaction with POPG cofactor unmasks a PrP(C) binding site, leading to PrP(Int1) aggregation.

Proteome and system ontology of hemorrhagic shock: exploring early constitutive changes in postshock mesenteric lymph.

BACKGROUND: Postshock mesenteric lymph (PSML) is the mechanistic link between splanchnic ischemia reperfusion (IR) and remote organ injury. We hypothesize that an unbiased inspection of the proteome of PSML will reveal previously unrecognized aberrations in systems biology provoked by hemorrhage-induced mesenteric IR injury in vivo. METHODS: Shock was induced in male Sprague-Dawley rats by controlled hemorrhage, and the mesenteric duct was cannulated for lymph collection. Preshock and postshock lymph were collected for differential in-gel electrophoresis (DIGE)-based proteomics. Proteins that increased or decreased in relative concentration > or =1.5-fold were selected for trypsin digestion and analysis by mass spectrometry (MS). RESULTS: Evidence of tissue injury was detected by an increase in cell/tissue proteins in PSML. Components of coagulation were depleted, whereas products of hemolysis were increased. Haptoglobin was decreased, which supports an early postshock hemolytic process. Interestingly, several protective protease inhibitors were decreased in PSML. The unexpected findings were an increase in alpha-enolase (a key glycolitic enzyme and cell-surface plasminogen binding receptor, +2.4-fold change) and increased major urinary protein (MUP, a sex-specific lipid-binding protein, +17.1-fold change) in PSML. CONCLUSION: A proteomic evaluation of PSML revealed evidence of several shock-associated processes: protein release from tissue injury, depletion of coagulation factors and evidence of hemolysis, depletion of protective protease inhibitors, and an increase in abundance of lipid carriers. These results suggest that constitutive changes in the proteome of PSML may provide novel insights into the complex pathophysiology of postshock systems biology.