A Single Kinase Generates the Majority of the Secreted Phosphoproteome.

The existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.

Structure-activity relationship of dihydropyridines for rhabdomyosarcoma.

Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of ∼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.

Overnight attending radiologist coverage decreases imaging-related emergency department recalls by at least 90.

PURPOSE: Benefits of overnight attending radiologist final reports are debated, often stating low resident discrepancy rates, usually assessed retrospectively. The objective of this study was to assess the impact of overnight final reporting on the recall rates for patients in the emergency department (ED) receiving overnight imaging. METHODS: Retrospective matched cohorts of two separate years prior (prior-16 and prior-17) and 1 year after (post-18) introduction of overnight attending radiologist final reporting. Patients receiving imaging between 22:00 and 07:00 h and returned to ED within 48 h of initial visit discharge were electronically identified. String matching identified return visits possibly related to imaging completed on first visit. Identified return visit notes were scored by three observers individually. Unclear and discrepant cases were resolved by consensus meeting, using full patient charts where needed. Incidences were provided and logistic regression analysis defined if coverage model was a predictor for recall. Odds ratios were calculated. RESULTS: ED patient count with imaging completed overnight in prior-16 was 9200, in prior-17 was 9543, and in post-18 was 9992. The number of overnight imaging studies performed was respectively 13,883, 14,463, and 15,112. Imaging-related ED recalls were respectively 54, 61, and 7, a decrease with the new coverage model of 89% to true and at least 90% of expected recalls.Logistic regression demonstrated that coverage model was a significant predictor of ED recalls with chi-square of 59.86 and p < 0.001, an R2 of 0.03 (Hosmer and Lemeshow). Compared to post-18, ED patients had an odds ratio of 8.42 (prior-16) and 9.18 (prior-17) to be called back to ED. CONCLUSION: Overnight final reporting significantly decreases ED recalls for patients receiving diagnostic imaging overnight. While numbers are low even prior to rollout, the number should be minimized wherever possible to diminish patient anxiety and discomfort, reduce ED overcrowding and expedite definitive management. KEY MESSAGES/WHAT THIS PAPER ADDS: Section 1: What is already known on this subject • Radiology resident preliminary report discrepancy rates are low. • Overnight attending radiologist coverage is a model increasingly applied in academic and large non-academic centers. • Patient recalls to the ED are a burden to the patient and impact patient throughput in (over)crowded EDs. Section 2: What this study adds • First study to look at the impact of overnight attending final reports on the recall rate for ED patients with overnight imaging performed. • While absolute numbers are low, there is a significant decrease in patients returning to ED for imaging related issues after introducing overnight attending coverage. • Resident autonomy can be preserved and training enhanced while increasing patient safety and comfort.

Evaluation of Bowel and Mesentery in Abdominal Trauma.

Since the advent of multidetecter computed tomography (CT), radiologist sensitivity in detection of traumatic bowel and mesenteric abnormalities has significantly improved. Although several CT signs have been described to identify intestinal injury, accurate interpretation of these findings can remain challenging. Early detection of bowel and mesenteric injury is important as it alters patient management, disposition, and follow-up. This article reviews the common imaging findings of traumatic small bowel and mesenteric injury.

Phosphorylation of spore coat proteins by a family of atypical protein kinases.

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

Comparative analysis of Histophilus somni immunoglobulin-binding protein A (IbpA) with other fic domain-containing enzymes reveals differences in substrate and nucleotide specificities.

A new family of adenylyltransferases, defined by the presence of a Fic domain, was recently discovered to catalyze the addition of adenosine monophosphate (AMP) to Rho GTPases (Yarbrough, M. L., Li, Y., Kinch, L. N., Grishin, N. V., Ball, H. L., and Orth, K. (2009) Science 323, 269-272; Worby, C. A., Mattoo, S., Kruger, R. P., Corbeil, L. B., Koller, A., Mendez, J. C., Zekarias, B., Lazar, C., and Dixon, J. E. (2009) Mol. Cell 34, 93-103). This adenylylation event inactivates Rho GTPases by preventing them from binding to their downstream effectors. We reported that the Fic domain(s) of the immunoglobulin-binding protein A (IbpA) from the pathogenic bacterium Histophilus somni adenylylates mammalian Rho GTPases, RhoA, Rac1, and Cdc42, thereby inducing host cytoskeletal collapse, which allows H. somni to breach alveolar barriers and cause septicemia. The IbpA-mediated adenylylation occurs on a functionally critical tyrosine in the switch 1 region of these GTPases. Here, we conduct a detailed characterization of the IbpA Fic2 domain and compare its activity with other known Fic adenylyltransferases, VopS (Vibrio outer protein S) from the bacterial pathogen Vibrio parahaemolyticus and the human protein HYPE (huntingtin yeast interacting protein E; also called FicD). We also included the Fic domains of the secreted protein, PfhB2, from the opportunistic pathogen Pasteurella multocida, in our analysis. PfhB2 shares a common domain architecture with IbpA and contains two Fic domains. We demonstrate that the PfhB2 Fic domains also possess adenylyltransferase activity that targets the switch 1 tyrosine of Rho GTPases. Comparative kinetic and phylogenetic analyses of IbpA-Fic2 with the Fic domains of PfhB2, VopS, and HYPE reveal important aspects of their specificities for Rho GTPases and nucleotide usage and offer mechanistic insights for determining nucleotide and substrate specificities for these enzymes. Finally, we compare the evolutionary lineages of Fic proteins with those of other known adenylyltransferases.

Structural insights into glucan phosphatase dynamics using amide hydrogen-deuterium exchange mass spectrometry.

Laforin and starch excess 4 (SEX4) are founding members of a class of phosphatases that dephosphorylate phosphoglucans. Each protein contains a carbohydrate binding module (CBM) and a dual-specificity phosphatase (DSP) domain. The gene encoding laforin is mutated in a fatal neurodegenerative disease called Lafora disease (LD). In the absence of laforin function, insoluble glucans that are hyperphosphorylated and exhibit sparse branching accumulate. It is hypothesized that these accumulations trigger the neurodegeneration and premature death of LD patients. We recently demonstrated that laforin removes phosphate from phosphoglucans and hypothesized that this function inhibits insoluble glucan accumulation. Loss of SEX4 function in plants yields a similar cellular phenotype; an excess amount of insoluble, hyperphosphorylated glucans accumulates in cells. While multiple groups have shown that these phosphatases dephosphorylate phosphoglucans, there is no structure of a glucan phosphatase and little is known about the mechanism whereby they perform this action. We utilized hydrogen-deuterium exchange mass spectrometry (DXMS) and structural modeling to probe the conformational and structural dynamics of the glucan phosphatase SEX4. We found that the enzyme does not undergo a global conformational change upon glucan binding but instead undergoes minimal rearrangement upon binding. The CBM has improved protection from deuteration when bound to glucans, confirming its role in glucan binding. More interestingly, we identified structural components of the DSP that also have improved protection from deuteration upon glucan addition. To determine the position of these regions, we generated a homology model of the SEX4 DSP. The homology model shows that all of these regions are adjacent to the DSP active site. Therefore, our results suggest that these regions of the DSP participate in the presentation of the phosphoglucan to the active site and provide the first structural analysis and mode of action of this unique class of phosphatases.

The microprotein Minion controls cell fusion and muscle formation.

Although recent evidence has pointed to the existence of small open reading frame (smORF)-encoded microproteins in mammals, their function remains to be determined. Skeletal muscle development requires fusion of mononuclear progenitors to form multinucleated myotubes, a critical but poorly understood process. Here we report the identification of Minion (microprotein inducer of fusion), a smORF encoding an essential skeletal muscle specific microprotein. Myogenic progenitors lacking Minion differentiate normally but fail to form syncytial myotubes, and Minion-deficient mice die perinatally and demonstrate a marked reduction in fused muscle fibres. The fusogenic activity of Minion is conserved in the human orthologue, and co-expression of Minion and the transmembrane protein Myomaker is sufficient to induce cellular fusion accompanied by rapid cytoskeletal rearrangement, even in non-muscle cells. These findings establish Minion as a novel microprotein required for muscle development, and define a two-component programme for the induction of mammalian cell fusion. Moreover, these data also significantly expand the known functions of smORF-encoded microproteins.