Mutations in ACY1, the gene encoding aminoacylase 1, cause a novel inborn error of metabolism

N-terminal acetylation of proteins is a widespread and highly conserved process. Aminoacylase 1 (ACY1; EC 3.5.14) is the most abundant of the aminoacylases, a class of enzymes involved in hydrolysis of N-acetylated proteins. Here, we present four children with genetic deficiency of ACY1. They were identified through organic acid analyses using gas chromatography-mass spectrometry, revealing increased urinary excretion of several N-acetylated amino acids, including the derivatives of methionine, glutamic acid, alanine, leucine, glycine, valine, and isoleucine. Nuclear magnetic resonance spectroscopy analysis of urine samples detected a distinct pattern of N-acetylated metabolites, consistent with ACY1 dysfunction. Functional analyses of patients' lymphoblasts demonstrated ACY1 deficiency. Mutation analysis uncovered recessive loss-of-function or missense ACY1 mutations in all four individuals affected. We conclude that ACY1 mutations in these children led to functional ACY1 deficiency and excretion of N-acetylated amino acids. Questions remain, however, as to the clinical significance of ACY1 deficiency. The ACY1-deficient individuals were ascertained through urine metabolic screening because of unspecific psychomotor delay (one subject), psychomotor delay with atrophy of the vermis and syringomyelia (one subject), marked muscular hypotonia (one subject), and follow-up for early treated biotinidase deficiency and normal clinical findings (one subject). Because ACY1 is evolutionarily conserved in fish, frog, mouse, and human and is expressed in the central nervous system (CNS) in human, a role in CNS function or development is conceivable but has yet to be demonstrated. Thus, at this point, we cannot state whether ACY1 deficiency has pathogenic significance with pleiotropic clinical expression or is simply a biochemical variant. Awareness of this new genetic entity may help both in delineating its clinical significance and in avoiding erroneous diagnoses.     

The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.     

The unfolded protein response is shaped by the NMD pathway

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD''s ability to regulate normal mRNAs.     

The Beauty is a beast: Does leachate from the invasive terrestrial plant Impatiens glandulifera affect aquatic food webs?

Invasive alien species are a major threat to ecosystems. Invasive terrestrial plants can produce allelochemicals which suppress native terrestrial biodiversity. However, it is not known if leached allelochemicals from invasive plants growing in riparian zones, such as Impatiens glandulifera, also affect freshwater ecosystems. We used mesocosms and laboratory experiments to test the impact of I. glandulifera on a simplified freshwater food web. Our mesocosm experiments show that leachate from I. glandulifera significantly reduced population growth rate of the water flea Daphnia magna and the green alga Acutodesmus obliquus, both keystone species of lakes and ponds. Laboratory experiments using the main allelochemical released by I. glandulifera, 2‐methoxy‐1,4‐naphthoquinone, revealed negative fitness effects in D. magna and A. obliquus. Our findings show that allelochemicals from I. glandulifera not only reduce biodiversity in terrestrial habitats but also pose a threat to freshwater ecosystems, highlighting the necessity to incorporate cross‐ecosystem effects in the risk assessment of invasive species.     

Geographic Variation in Male Sexual Signals in Strawberry Poison Frogs (Dendrobates pumilio)

In this paper, we compare the advertisement calls of 207 neotropical strawberry poison frogs (Dendrobates pumilio) collected in 21 localities along a transect from northern Costa Rica to western Panama. Populations varied most in call duration and call rate, while pulse rate and duty cycle were less variable. Multivariate analyses showed that call variation followed a cline with higher call rates, shorter calls, lower duty cycles and higher pulse rates in the southeast. Body size decreased towards the southeast and explained most variation in dominant frequency, as well as some residual variation in call rate. We conclude that a combination of geography and morphology is largely responsible for call variation within this species. Two inferred bio‐acoustic groups were roughly in accordance with two genetic groups, geographically separated in central Costa Rica. However, genetic distances among populations did not co‐vary with call dissimilarity after correction for geographic distances. Thus, differences in calls between genetic groups are probably mainly a result of clinal variation. These findings agree with the general observation that bio‐acoustic variation is often not (highly) associated with genetic divergence. Moreover, colour polymorphism observed among Panamanian populations was not reflected in a higher variability in call parameters relative to the monomorphic Costa Rican populations.     

Unusual evolution of a catalytic core element in CCA-adding enzymes

CCA-adding enzymes are polymerases existing in two distinct enzyme classes that both synthesize the C-C-A triplet at tRNA 3′-ends. Class II enzymes (found in bacteria and eukaryotes) carry a flexible loop in their catalytic core required for switching the specificity of the nucleotide binding pocket from CTP- to ATP-recognition. Despite this important function, the loop sequence varies strongly between individual class II CCA-adding enzymes. To investigate whether this loop operates as a discrete functional entity or whether it depends on the sequence context of the enzyme, we introduced reciprocal loop replacements in several enzymes. Surprisingly, many of these replacements are incompatible with enzymatic activity and inhibit ATP-incorporation. A phylogenetic analysis revealed the existence of conserved loop families. Loop replacements within families did not interfere with enzymatic activity, indicating that the loop function depends on a sequence context specific for individual enzyme families. Accordingly, modeling experiments suggest specific interactions of loop positions with important elements of the protein, forming a lever-like structure. Hence, although being part of the enzyme’s catalytic core, the loop region follows an extraordinary evolutionary path, independent of other highly conserved catalytic core elements, but depending on specific sequence features in the context of the individual enzymes.     

Novel bacteriochlorophyll <Emphasis Type="BoldItalic">e</Emphasis> structures and species-specific variability of pigment composition in green sulfur bacteria

The relative composition of bacteriochlorophyll (BChl) homologs in five different strains of brown-colored green sulfur bacteria was investigated by HPLC-MS/MS and NMR analyses. In addition, the effect of incubation light intensities on homolog distribution was studied in one of the strains (strain Dagow III). A total of 23 different BChl e structures were detected and comprise four homologous porphyrin ring systems and eight different esterifying alcohols. Several BChl e structures are novel. These include a C-8 ethyl, C-12 methyl [E, M] BChl e(F) homolog which was identified by (1)H-NMR analyses of the isolated, main farnesyl homologs (BChl e(F)). In addition, five previously unknown homolog series with dodecanol, pentadecenol, tetradecanol, hexadecenol and phytol as the esterifying alcohols were detected. The composition of BChl e homologs from the five strains of green sulfur bacteria differed with respect to the relative abundance of the homologs (BChl e(F) : 25.6-67.0% of total BChl e content in stationary cultures). In strain Dagow III, the abundance of BChl e(F) homologs decreased upon entry into the stationary phase. In all free-living strains, the abundance of BChl e(F) was increased when the relative carotenoid content was low. The present results provide a detailed picture of pigment composition in chlorosomes and thus will help to elucidate their structure and function. Furthermore, the newly discovered BChl e molecules are valuable biomarkers for the study of the occurrence and metabolism of green sulfur bacteria in past and present ecosystems.     

c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13-22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.