Craig D. Thulin

Activation of the furin endoprotease is a multiple‐step process: requirements for acidification and internal propeptide cleavage

Activation of furin requires autoproteolytic cleavage of its 83‐amino acid propeptide at the consensus furin site, Arg‐Thr‐Lys‐Arg107↓. This RER‐localized cleavage is necessary, but not sufficient, for enzyme activation. Rather, full activation of furin requires exposure to, and correct routing within, the TGN/endosomal system. Here, we identify the steps in addition to the initial propeptide cleavage necessary for activation of furin. Exposure of membrane preparations containing an inactive RER‐localized soluble furin construct to either: (i) an acidic and calcium‐containing environment characteristic of the TGN; or (ii) mild trypsinization at neutral pH, resulted in the activation of the endoprotease. Taken together, these results suggest that the pH drop facilitates the removal of a furin inhibitor. Consistent with these findings, following cleavage in the RER, the furin propeptide remains associated with the enzyme and functions as a potent inhibitor of the endoprotease. Co‐immunoprecipitation studies coupled with analysis by mass spectrometry show that release of the propeptide at acidic pH, and hence activation of furin, requires a second cleavage within the autoinhibitory domain at a site containing a P6 arginine (‐Arg70‐Gly‐Val‐Thr‐Lys‐Arg75↓‐). The significance of this cleavage in regulating the compartment‐specific activation of furin, and the relationship of the furin activation pathway to those of other serine endoproteases are discussed.

Noncontact dipole effects on channel permeation. I. Experiments with (5F-indole)Trp13 gramicidin A channels.

Gramicidin A (gA), with four Trp residues per monomer, has an increased conductance compared to its Phe replacement analogs. When the dipole moment of the Trp13 side chain is increased by fluorination at indole position 5 (FgA), the conductance is expected to increase further. gA and FgA conductances to Na+, K+, and H+ were measured in planar diphytanoylphosphatidylcholine (DPhPC) or glycerylmonoolein (GMO) bilayers. In DPhPC bilayers, Na+ and K+ conductances increased upon fluorination, whereas in GMO they decreased. The low ratio in the monoglyceride bilayer was not reversed in GMO-ether bilayers, solvent-inflated or -deflated bilayers, or variable fatty acid chain monoglyceride bilayers. In both GMO and DPhPC bilayers, fluorination decreased conductance to H+ but increased conductance in the mixed solution, 1 M KCl at pH 2.0, where K+ dominates conduction. Eadie-Hofstee plot slopes suggest similar destabilization of K+ binding in both lipids. Channel lifetimes were not affected by fluorination in either lipid. These observations indicate that fluorination does not change the rotameric conformation of the side chain. The expected difference in the rate-limiting step for transport through channels in the two bilayers qualitatively explains all of the above trends.

Regulatory interaction of phosducin-like protein with the cytosolic chaperonin complex

Phosducin and phosducin-like protein (PhLP) bind G protein βγ subunits and regulate their activity. This report describes a previously uncharacterized binding partner unique to PhLP that was discovered by coimmunoprecipitation coupled with mass spectrometric identification. Chaperonin containing tailless complex polypeptide 1 (CCT), a cytosolic chaperone responsible for the folding of many cellular proteins, binds PhLP with a stoichiometry of one PhLP per CCT complex. Unlike protein-folding substrates of CCT, which interact only in their nonnative conformations, PhLP binds in its native state. Native PhLP competes directly for binding of protein substrates of CCT and thereby inhibits CCT activity. Overexpression of PhLP inhibited the ability of CCT to fold newly synthesized β-actin by 80%. These results suggest that the interaction between PhLP and CCT may be a means to regulate CCT-dependent protein folding or alternatively, to control the availability of PhLP to modulate G protein signaling.

Mechanism of Assembly of G Protein βγ Subunits by Protein Kinase CK2-phosphorylated Phosducin-like Protein and the Cytosolic Chaperonin Complex

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein βγ subunit complex (Gβγ) that has been recently shown to catalyze the formation of the Gβγ dimer from its nascent polypeptides. Phosphorylation of PhLP at one or more of three consecutive serines (Ser-18, Ser-19, and Ser-20) is necessary for Gβγ dimer formation and is believed to be mediated by the protein kinase CK2. Moreover, several lines of evidence suggest that the cytosolic chaperonin complex (CCT) may work in concert with PhLP in the Gβγ-assembly process. The results reported here delineate a mechanism for Gβγ assembly in which a stable ternary complex is formed between PhLP, the nascent Gβ subunit, and CCT that does not include Gγ. PhLP phosphorylation permits the release of a PhLP·Gβ intermediate from CCT, allowing Gγ to associate with Gβ in this intermediate complex. Subsequent interaction of Gβγ with membranes releases PhLP for another round of assembly.

Modulation of the G Protein Regulator Phosducin by Ca2+/Calmodulin-dependent Protein Kinase II Phosphorylation and 14-3-3 Protein Binding

Phototransduction is a canonical G protein-mediated cascade of retinal photoreceptor cells that transforms photons into neural responses. Phosducin (Pd) is a Gβγ-binding protein that is highly expressed in photoreceptors. Pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. Dephosphorylated Pd binds Gβγ with high affinity and inhibits the interaction of Gβγ with Gα or other effectors, whereas phosphorylated Pd does not. These results have led to the hypothesis that Pd down-regulates the light response. Consequently, it is important to understand the mechanisms of regulation of Pd phosphorylation. We have previously shown that phosphorylation of Pd by cAMP-dependent protein kinase moderately inhibits its association with Gβγ. In this study, we report that Pd was rapidly phosphorylated by Ca2+/calmodulin-dependent kinase II, resulting in 100-fold greater inhibition of Gβγ binding than cAMP-dependent protein kinase phosphorylation. Furthermore, Pd phosphorylation by Ca2+/calmodulin-dependent kinase II at Ser-54 and Ser-73 led to binding of the phosphoserine-binding protein 14-3-3. Importantly, in vivodecreases in Ca2+ concentration blocked the interaction of Pd with 14-3-3, indicating that Ca2+ controls the phosphorylation state of Ser-54 and Ser-73 in vivo. These results are consistent with a role for Pd in Ca2+-dependent light adaptation processes in photoreceptor cells and also suggest other possible physiological functions.

Mechanism of assembly of G protein βγ subunits by CK2-phosphorylated phosducin-like protein and the cytosolic chaperonin complex

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein βγ subunit complex (Gβγ) that has been recently shown to catalyze the formation of the Gβγ dimer from its nascent polypeptides. Phosphorylation of PhLP at one or more of three consecutive serines (Ser-18, Ser-19, and Ser-20) is necessary for Gβγ dimer formation and is believed to be mediated by the protein kinase CK2. Moreover, several lines of evidence suggest that the cytosolic chaperonin complex (CCT) may work in concert with PhLP in the Gβγ-assembly process. The results reported here delineate a mechanism for Gβγ assembly in which a stable ternary complex is formed between PhLP, the nascent Gβ subunit, and CCT that does not include Gγ. PhLP phosphorylation permits the release of a PhLP·Gβ intermediate from CCT, allowing Gγ to associate with Gβ in this intermediate complex. Subsequent interaction of Gβγ with membranes releases PhLP for another round of assembly.

Regulation of Angiotensin II-induced G Protein Signaling by Phosducin-like Protein

Phosducin-like protein (PhLP) is a broadly expressed member of the phosducin (Pd) family of G protein βγ subunit (Gβγ)-binding proteins. Though PhLP has been shown to bind Gβγ in vitro, little is known about its physiological function. In the present study, the effect of PhLP on angiotensin II (Ang II) signaling was measured in Chinese hamster ovary cells expressing the type 1 Ang II receptor and various amounts of PhLP. Up to 3.6-fold overexpression of PhLP had no effect on Ang II-stimulated inositol trisphosphate (IP3) formation, whereas further increases caused an abrupt decrease in IP3 production with half-maximal inhibition occurring at 6-fold PhLP overexpression. This threshold level for inhibition corresponds to the cellular concentration of cytosolic chaperonin complex, a recently described binding partner that preferentially binds PhLP over Gβγ. Results of pertussis toxin sensitivity, GTPγS binding, and immunoprecipitation experiments suggest that PhLP inhibits phospholipase Cβ activation by dual mechanisms: (i) steric blockage of Gβγ activation of PLCβ and (ii) interference with Gβγ-dependent cycling of Gqα by the receptor. These results suggest that G protein signaling may be regulated through controlling the cellular concentration of free PhLP by inducing its expression or by regulating its binding to the chaperonin.

An integrated serum proteomic approach capable of monitoring the low molecular weight proteome with sequencing of intermediate to large peptides

The low-abundance, low molecular weight serum proteome has high potential for the discovery of new biomarkers using mass spectrometry (MS). Because the serum proteome is large and complex, defining relative quantitative differences for a molecular species between comparison groups requires an approach with robust separation capability, high sensitivity, as well as high mass resolution. Capillary liquid chromatography (cLC)/MS provides both the necessary separation technique and the sensitivity to observe many low-abundance peptides. Subsequent identification of potential serum peptide biomarkers observed in the cLC/MS step can in principle be accomplished by in series cLC/MS/MS without further sample preparation or additional instrumentation. In this report a novel cLC/MS/MS method for peptide sequencing is described that surpasses previously reported size limits for amino acid sequencing accomplished by collisional fragmentation using a tandem time-of-flight MS instrument. As a demonstration of the approach, two low-abundance peptides with masses of approximately 4000-5000 Da were selected for MS/MS sequencing. The multi-channel analyzer (MCA) was used in a novel way that allowed for summation of 120 fragmentation spectra for each of several customized collision energies, providing more thorough fragmentation coverage of each peptide with improved signal to noise. The peak list from this composite analysis was submitted to Mascot for identification. The two index peptides, 4279 Da and 5061 Da, were successfully identified. The peptides were a 39 amino acid immunoglobulin G heavy chain variable region fragment and a 47 amino acid fibrin alpha isoform C-terminal fragment. The method described here provides the ability both to survey thousands of serum molecules and to couple that with markedly enhanced cLC/MS/MS peptide sequencing capabilities, providing a promising technique for serum biomarker discovery.