Analysis of Site-Specific Phosphorylation of PTEN by Using Enzyme-Catalyzed Expressed Protein Ligation

The activity and localization of PTEN, a tumor suppressor lipid phosphatase that converts the phospholipid PIP3 to PIP2, is governed in part by phosphorylation on a cluster of four Ser and Thr residues near the C terminus. Prior enzymatic characterization of the four monophosphorylated (1p) PTENs by using classical expressed protein ligation (EPL) was complicated by the inclusion of a non-native Cys at the ligation junction (aa379), which may alter the properties of the semisynthetic protein. Here, we apply subtiligase-mediated EPL to create wt 1p-PTENs. These PTENs are more autoinhibited than previously appreciated, consistent with the role of Tyr379 in driving autoinhibition. Alkaline phosphatase sensitivity analysis revealed that these autoinhibited 1p conformations are kinetically labile. In contrast to the Cys mutant 1p-PTENs, which are poorly recognized by an anti-phospho-PTEN antibody, three of the four wt 1p-PTENs are recognized by a commonly used anti-phospho-PTEN antibody.     

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein–protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling‐transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C‐terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand‐binding site of the second PDZ domain of PSD‐95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the β1–β2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.     

Facile One‐Step Assembly of Bona Fide SUMO Conjugates by Chemoenzymatic Ligation

The post‐translational conjugation of the small ubiquitin‐like modifiers (SUMOs) to target proteins occurs through a complex machinery that involves sequential action of at least three enzymes. SUMOylation performs crucial regulatory functions in several cellular processes. The availability of well‐defined SUMO conjugates is necessary for untangling the mechanism of SUMOylation. However, assembly of homogeneous SUMO conjugates represents a challenge because of the multi‐step synthesis involved and the unwieldiness of the reconstituted biosynthetic systems. Here we describe a simple one‐step chemoenzymatic strategy for conjugating engineered SUMO (eSUMO) proteins to a prefabricated isopeptide‐linked SUMO target peptide. Notably, the eSUMOs were efficiently recognized by the enzymes of the SUMOylation machinery and the SUMO conjugates served as bona fide substrates for DeSUMOylating enzymes.     

SynBio-SynChem Approaches to Diversifying the Pacidamycins through the Exploitation of an Observed Pictet-Spengler Reaction

A nonenzymatic Pictet-Spengler reaction has been postulated to give rise to a subset of naturally occurring uridyl peptide antibiotics (UPAs). Here, using a combination of strain engineering and synthetic chemistry, we demonstrate that Pictet-Spengler chemistry may be employed to generate even greater diversity in the UPAs. We use an engineered strain to afford access to meta-tyrosine containing pacidamycin 4. Pictet-Spengler diversification of this compound using a small series of aryl-aldehydes was achieved with some derivatives affording remarkable diastereomeric control.     

Towards Dissecting the Mechanism of Protein Phosphatase-1 Inhibition by Its C-Terminal Phosphorylation

Phosphoprotein phosphatase-1 (PP1) is a key player in the regulation of phospho-serine (pSer) and phospho-threonine (pThr) dephosphorylation and is involved in a large fraction of cellular signaling pathways. Aberrant activity of PP1 has been linked to many diseases, including cancer and heart failure. Besides a well-established activity control by regulatory proteins, an inhibitory function for phosphorylation (p) of a Thr residue in the C-terminal intrinsically disordered tail of PP1 has been demonstrated. The associated phenotype of cell-cycle arrest was repeatedly proposed to be due to autoinhibition of PP1 through either conformational changes or substrate competition. Here, we use PP1 variants created by mutations and protein semisynthesis to differentiate between these hypotheses. Our data support the hypothesis that pThr exerts its inhibitory function by mediating protein complex formation rather than by a direct mechanism of structural changes or substrate competition.     

Engineered Sortases in Peptide and Protein Chemistry

The transpeptidase sortase A of Staphylococcus aureus (Sa-SrtA) is a valuable tool in protein chemistry. The native enzyme anchors surface proteins containing a highly conserved LPxTG sorting motif to a terminal glycine residue of the peptidoglycan layer in Gram-positive bacteria. This reaction is exploited for sortase-mediated ligation (SML), allowing the site-specific linkage of synthetic peptides and recombinant proteins by a native peptide bond. However, the moderate catalytic efficiency and specificity of Sa-SrtA fueled the development of new biocatalysts for SML, including the screening of sortase A variants form microorganisms other than S. aureus and the directed protein evolution of the Sa-SrtA enzyme itself. Novel display platforms and screening formats were developed to isolate sortases with altered properties from mutant libraries. This yielded sortases with strongly enhanced catalytic activity and enzymes recognizing new sorting motifs as substrates. This minireview focuses on recent advances in the field of directed sortase evolution and applications of these tailor-made enzymes in biochemistry.     

The semisynthesis of octadeutero-PheB1-octadeutero-ValB2]-procine insulin and its characterization by mass spectrometry

Insulin analogues labelled with stable isotopes (e.g. deuterium,¹⁸O, ¹⁵N, etc.) are authentic (the native structure is rigorouslymaintained), non-radioactive (preferred for injection into man)and can easily be distinguished from endogenous insulin by massspectrometry by virtue of their molecular masses. Appropriatecombinations of amino-protecting groups (methylsulphonylethyloxycarbonyland t-butoxy carbonyl), Edman degradation and chemical couplingwere used to produce [octadeutero-Phe^B1]-porcine insulin and[octadeutero-Phe^B1-octadeutero-Val^B2]-porcine insulin. The analogueswere characterized by electrospray ionization mass spectrometry.Standard mixtures of labelled and unlabelled insulins were successfullystudied by mass spectrometry. Isotope dilution mass spectrometrycould therefore provide a useful direct measure of insulin undertrue physiological conditions, without many of the drawbacksof existing methods. In this regard, the analogue with 16 deuteriumswas more suitable than the octadeuterated analogue, since thegreater mass difference between the labelled and unlabelledforms enabled a lower mass spectrometric resolution to be used,resulting in higher sensitivity