Ubiquitin phosphorylated at Ser57 hyper-activates parkin.

Malfunction of the ubiquitin (Ub) E3 ligase, parkin, leads to defects in mitophagy and protein quality control linked to Parkinson's disease. Parkin activity is stimulated by phosphorylation of Ub at Ser65 (pUbS65). Since the upstream kinase is only known for Ser65 (PINK1), the biochemical function of other phosphorylation sites on Ub remain largely unknown. We used fluorescently labelled and site-specifically phosphorylated Ub substrates to quantitatively relate the position and stoichiometry of Ub phosphorylation to parkin activation. Fluorescence measurements show that pUbS65-stimulated parkin is 5-fold more active than auto-inhibited and un-stimulated parkin, which catalyzes a basal level of auto-ubiquitination. We consistently observed a low but detectable level of parkin activity with pUbS12. Strikingly, pUbS57 hyper-activates parkin, and our data demonstrate that parkin is able to selectively synthesize poly-pUbS57 chains, even when 90% of the Ub in the reaction is un-phosphorylated. We further found that parkin ubiquitinates its physiological substrate Miro-1 with chains solely composed of pUbS65 and more efficiently with pUbS57 chains. Parkin hyper-activation by pUbS57 demonstrates the first PINK1-independent route to active parkin, revealing the roles of multiple ubiquitin phosphorylation sites in governing parkin stimulation and catalytic activity. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Generation of phospho-ubiquitin variants by orthogonal translation reveals codon skipping.

The activity of the Parkinson's disease-linked E3 ligase parkin is stimulated by phosphorylation at ubiquitin Ser65 (pUb(S65) ). The role of other ubiquitin phospho-sites and their kinases are unknown. We produced pUb variants (pS7, pS12, pS20, pS57, pS65) by genetically encoding phosphoserine with the UAG codon. In release factor-deficient Escherichia coli (ΔRF1), intended to enhance UAG read-through, we discovered ubiquitin variants lacking the UAG-encoded residue, demonstrating previously undocumented +3 frame shifting. We successfully purified each pUb variant from mistranslated products. While pUb(S20) failed to stimulate parkin, parkin was partially active with pUb(S12) . We observed significant ubiquitination when pUb(S65) was the sole substrate.

Ureteroscope cleaning and sterilization by the urology operating room team: the effect on repair costs.

BACKGROUND AND PURPOSE: Flexible ureteroscopes are fragile devices, and the costs associated with their repair and replacement can be considerable. Although surgical use can degrade ureteroscope function, the cleaning and sterilization process can also cause great damage. We performed a study to define the effect of having the urology nursing staff process and sterilize all ureteroscopes, rather than the central processing core; the total repair cost and cost per use were analyzed. MATERIALS AND METHODS: From April 2007 to March 2008, all ureteroscopes were processed by the urology nursing staff. We analyzed the average cost per use as a measure of the effectiveness of this strategy. For all endoscopic stone removal cases, a flexible ureteroscope is opened onto the operative field; therefore, after every endoscopic procedure, the flexible ureteroscope needs processing and sterilizing. The number of times each ureteroscope was processed and the type and cost of repairs were recorded. RESULTS: From April 2007 to March 2008, 11 ureteroscopes were processed 478 times; average uses before repair was 28.1. Seven ureteroscopes were returned for repair because of: loss of deflection (2); loss of fiberoptic bundles (2); failed leak test (3). No ureteroscope damage was because of processing. The total repair cost in this 12-month period was $57,664.50. Amortizing repair costs over use gives a value of $120.63 cost per use. CONCLUSIONS: Training the urology nursing staff to clean and sterilize ureteroscopes is a reasonable means to reduce processing-related damages.