Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay.

Chemical details of intramembrane proteolysis remain elusive despite its prevalence throughout biology. We developed a FRET peptide assay for the intramembrane aspartyl protease (IAP) from Methanoculleus marisnigri JR1 in combination with quantitative mass spectrometry cleavage site analysis. IAP can hydrolyze the angiotensinogen sequence, a substrate for the soluble aspartyl protease renin, at a predominant cut site, His-Thr. Turnover is slow (min(-1) × 10(-3)), affinity and Michaelis constant (Km) values are in the low micromolar range, and both catalytic rates and cleavage sites are the same in detergent as reconstituted into bicelles. Three well-established, IAP-directed inhibitors were directly confirmed as competitive, albeit with modest inhibitor constant (Ki) values. Partial deletion of the first transmembrane helix results in a biophysically similar but less active enzyme than full-length IAP, indicating a catalytic role. Our study demonstrates previously unappreciated similarities with soluble aspartyl proteases, provides new biochemical features of IAP and inhibitors, and offers tools to study other intramembrane protease family members in molecular detail.

Rapid and effective removal of perfluorooctanoic acid from proteomics samples.

We recently demonstrated that perfluorooctanoic acid (PFOA), a volatile surfactant, is as effective as sodium dodecyl sulfate at solubilizing the membrane proteins. PFOA can be removed by repeated evaporation prior to mass spectrometry analysis. However, the removal of PFOA by evaporation is a lengthy process that takes approximately 6 h. Toward the goal of decreasing the length of time required to remove PFOA from protein digests, we tested the efficiency of PFOA removal and subsequent peptide recovery using strong cation exchange (SCX) chromatography, hydrophilic interaction chromatography (HILIC), fluorous solid phase extraction (FSPE), and anion exchange (ANX) chromatography. We found that all these chromatographic techniques except ANX chromatography remove PFOA thoroughly from protein digest. Peptide recovery rates from the SCX chromatography varied widely; nonacidic peptides were recovered at a rate of up to 95%, while acidic peptides were recovered at a rate of less than 10%. On the other hand, acidic peptides were recovered well from HILIC, while peptides whose pIs are greater than 6 were recovered poorly. Peptide recovery using FSPE was considerably lower, less than 10% for most of the peptides. These results indicate that the SCX and HILIC chromatography provide a more rapid alternative to the evaporation method for applications in which recovery of entire set of peptides is not required.

Streptomyces erythraeus trypsin inactivates α1-antitrypsin.

Streptomyces erythraeus trypsin (SET) is a serine protease that is secreted extracellularly by S. erythraeus. We investigated the inhibitory effect of α(1)-antitrypsin on the catalytic activity of SET. Intriguingly, we found that SET is not inhibited by α(1)-antitrypsin. Our investigations into the molecular mechanism underlying this observation revealed that SET hydrolyzes the Met-Ser bond in the reaction center loop of α(1)-antitrypsin. However, SET somehow avoids entrapment by α(1)-antitrypsin. We also confirmed that α(1)-antitrypsin loses its inhibitory activity after incubation with SET. Thus, our study demonstrates that SET is not only resistant to α(1)-antitrypsin but also inactivates α(1)-antitrypsin.

Purification and characterization of human nucleolar phosphoprotein 140 expressed in Escherichia coli.

Human nucleolar phosphoprotein 140, hNopp140, is one of the most highly phosphorylated mammalian proteins, which is involved in the biogenesis of nucleolus. It regulates the transcription of rDNA and has a tendency to bind to doxorubicin, which is widely used as an anti-cancer drug. The biochemical and biophysical property of hNopp140 has not been reported due to the fact that it is rather difficult to obtain protein in large enough quantity. In this paper, we report the cloning and overexpression of the soluble form of hNopp140 in Escherichia coli. The protein was purified to more than 90% homogeneity using hydroxyapatite and ion exchange chromatography. The purified protein can be extensively phosphorylated by casein kinase II and oligomerized into an insoluble aggregate in the presence of magnesium, carbonate, and fluoride ions.