Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser65 by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser65 phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin's ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser65 is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson's disease.

Human colostrum is an important source of protective, nutritional and developmental factors for the newborn. We have investigated the low abundance proteins in the aqueous phase of human colostrum, after depletion of the major proteins secretory IgA, lactoferrin, alpha-lactalbumin and HSA by immunoabsorption, using 2-D LC and gel-based proteomic methods. One hundred and fifty-one proteins were identified, 83 of which have not been previously reported in human colostrum, or milk. This is the first comprehensive proteomic analysis of human colostrum produced during the first 48 h of lactation.

HIF1A (hypoxia-inducible factor 1α) is the master regulator of the cellular response to hypoxia and is implicated in cancer progression. Whereas the regulation of HIF1A protein in response to oxygen is well characterized, less is known about the fate of HIF1A mRNA. In the present study, we have identified the pseudo-DUB (deubiquitinating enzyme)/deadenylase USP52 (ubiquitin-specific protease 52)/PAN2 [poly(A) nuclease 2] as an important regulator of the HIF1A-mediated hypoxic response. Depletion of USP52 reduced HIF1A mRNA and protein levels and resulted in reduced expression of HIF1A-regulated hypoxic targets due to a 3′-UTR (untranslated region)-dependent poly(A)-tail-length-independent destabilization in HIF1A mRNA. MS analysis revealed an association of USP52 with several P-body (processing body) components and we confirmed further that USP52 protein and HIF1A mRNA co-localized with cytoplasmic P-bodies. Importantly, P-body dispersal by knockdown of GW182 or LSM1 resulted in a reduction of HIF1A mRNA levels. These data uncover a novel role for P-bodies in regulating HIF1A mRNA stability, and demonstrate that USP52 is a key component of P-bodies required to prevent HIF1A mRNA degradation.

Previous investigations of bovine seminal plasma (BSP) have revealed the identities of the three major proteins, BSP-PDC109, BSP-A3 and BSP-30 kDa, which together constitute about half of the total protein, as well as about 30 of the minor proteins. Analyses of BSP by 2-DE have revealed about 250 protein spots, suggesting that much of the BSP proteome remains undescribed. In this study, BSP has been analyzed by 2-D LC-based and SDS-PAGE-based proteomic methods. Ninety-nine proteins were identified, including 49 minor proteins that have not previously been described in seminal plasma of any species.

Biotin is an essential vitamin in plants and mammals functioning as the carbon dioxide carrier within central lipid metabolism. Bacterial pimeloyl-CoA synthetase (BioW) acts as a highly specific substrate selection gate ensuring the integrity of the carbon chain in biotin synthesis.BioW catalyses the condensation of pimelic acid (C7 dicarboxylic acid) with CoASH in an ATP dependent manner to form pimeloyl-CoA, the first dedicated biotin building block.Multiple structures of Bacillus subtilis BioW together capture all three substrates as well as the intermediate pimeloyl-adenylate and product pyrophosphate (PP i ), and indicates that the enzyme uses an internal ruler to select the correct dicarboxylic acid substrate. Both the catalytic mechanism and the surprising stability of the adenylate intermediate were rationalized through site-directed mutagenesis. Building on this understanding, BioW was engineered to synthesise high value heptanoyl (C7) and octanoyl (C8) mono carboxylic acidCoA and C8 dicarboxylic-CoA products, highlighting the enzyme's synthetic potential.2

Hypoxemia is a defining feature of acute respiratory distress syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, yet the resulting tissue hypoxia, and its impact on immune responses, is often neglected. In the present study, we have shown that ARDS patients were hypoxemic and monocytopenic within the first 48 h of ventilation. Monocytopenia was also observed in mouse models of hypoxic acute lung injury, in which hypoxemia drove the suppression of type I interferon signaling in the bone marrow. This impaired monopoiesis resulted in reduced accumulation of monocyte-derived macrophages and enhanced neutrophil-mediated inflammation in the lung. Administration of colony-stimulating factor 1 in mice with hypoxic lung injury rescued the monocytopenia, altered the phenotype of circulating monocytes, increased monocyte-derived macrophages in the lung and limited injury. Thus, tissue hypoxia altered the dynamics of the immune response to the detriment of the host and interventions to address the aberrant response offer new therapeutic strategies for ARDS.

TamA is the adenylating enzyme that selects and activates fatty acids for tambjamine biosynthesis.

Non-protein-bound oligosaccharides are important bioactive components of cow milk, with potential human-health benefits such as stimulation of the growth of beneficial gut bacteria and defense against pathogens. In bovine milk, the majority of oligosaccharides are sialylated; 3'-sialyllactose (3'-N-acetylneuraminyl-D-lactose; 3'-SL) is the predominant sialylated oligosaccharide, followed by 6'-sialyllactose (6'-N-acetylneuraminyl-D-lactose; 6'-SL). Both 3'-SL and 6'-SL have antimicrobial activity. As bovine milk products such as infant formula can be an important component of the human diet, and the concentrations of 3'-SL and 6'-SL are lower in bovine milk compared with human milk, we aimed to identify cows that naturally produce higher concentrations of sialyllactose in their milk. Milk from such cows could be used to produce foods with an increased sialyllactose content, potentially providing increased health benefits. We speculated that cows overexpressing 3'-SL and 6'-SL would exist at low frequency in the population and, to allow their efficient identification, we developed a novel assay for 3'-SL and 6'-SL utilizing flow-injection analysis-mass spectrometry, which could be used for high-throughput analysis of milk samples. We then determined 3'-SL and 6'-SL concentrations in milk samples from 15,507 cows from Friesian, Jersey, and Friesian-Jersey crossbred animals. We found 329 cows with concentrations of 3'-SL or 6'-SL >2-fold higher than the mean, 26 cows with concentrations of 3'-SL or 6'-SL >3-fold higher than the mean, and 1 cow with concentrations of 3'-SL >4-fold higher than the mean. Although these outliers were observed across the 3 groups of cows, breed had a strong effect on mean 3'-SL and 6'-SL concentrations.