Gary L. Nelsestuen

Vitamin K-dependent proteins.

Vitamin K is required for the synthesis of gamma-carboxyglutamate (Gla) during postribosomal protein modification. Substrates include blood clotting proteins, bone proteins, cell signaling, and receptor proteins. In addition, Gla is a component of short toxin peptides from the marine snail Conus. Studies of structure-function relationships are the most advanced for the blood coagulation proteins. Reviews of vitamin K action and blood coagulation are presented. Special focus is on the structure-function role of Gla in blood coagulation and the impact of this amino acid on enzyme reaction kinetics. This amino acid forms calcium and membrane binding sites for these proteins. Two proposed mechanisms of protein-membrane attachment are reviewed. One involves membrane attachment by protein insertion into the hydrocarbon region of the membrane, while another considers attachment by specific interactions with phospholipid head groups. Membrane attachment generates the potential for several forms of nonclassical enzyme kinetic behaviors, all of which have been observed in vitro. For example, the reaction may be limited by properties of the enzyme active site, a condition that allows use of classic steady-state enzyme kinetic parameters. However, the reaction may be limited by substrate binding to the membrane, by substrate flux through solution, and/or by solvent flow rates across the membrane surface. These states provide special mechanisms that are not anticipated by classical steady-state kinetic derivations. They may be used to regulate coagulation in vivo. Overall, vitamin K research spans the spectrum of biological research and experience. Exciting new ideas and findings continue to emanate from vitamin K-related research.

Activation and regulation of protein Kinase C enzymes

Protein Kinase C (PKC) has been a principal regulatory enzyme whose function has been intensely investigated in the past decade. The primary features of this family of enzymes includes phosphorylation of serine and threonine residues located on basic proteins and peptides in a manner that is stimulated by calcium, phospholipid, and either diacylglycerol or phorbol esters. An additional intriguing feature of the enzyme is its ability to form two membrane-associated states, one of which is calcium dependent and reversible and the second is an irreversible complex which has the characteristics of an intrinsic membrane protein. Formation of the irreversible membrane-bound form is greatly facilitated by calcium and the tumor-promoting phorbol esters but does not appear to include covalent changes in the PKC structure. The intrinsic membrane-bound form is a very different enzyme in that its activity is no longer dependent on the other cofactors. It is proposed that formation of the irreversible membrane-bound form may be a mechanism for generating long-term cell regulation events where transient cell signals and second messengers induce long-term changes in the distribution of an enzyme in the cell. This property may be common to a number of regulatory proteins that are known to be distributed between the cytosol and membrane-fractions in the cell. Unfortunately, many problems have confronted study of PKC mechanism using thein vitro assay. This assay involves aggregation of the substrate, phospholipid, and enzyme to form a discontinuous mixture. Such as complex system prevents straightforward interpretation of enzyme kinetic data. Although many compounds affect thein vitro activity of PKC, most appear to accomplish this by relatively uninteresting mechanisms such as interference with the aggregation process. While some highly potent inhibitors undoubtedly interact directly with PKC, they also inhibit other enzymes and there are no entirely specific inhibitors of PKC known. Speculation on the possible roles of PKC in cell regulation are abundant and exciting. However, delineation of the regulatory roles of PKC may require another decade of intense effort.

Constitutive activity of membrane-inserted protein kinase C.

Incubation of purified protein kinase C (PKC) with phospholipid vesicles produced two populations of membrane-bound PKC: one population was dissociated by calcium chelation and the other was not. The second population appeared to be inserted into the membrane. The activity of membrane-inserted PKC was Ca2+-independent and was only modestly sensitive to phorbol esters. Insertion was caused by high calcium concentrations or by phorbol esters plus low calcium. These conditions correlated with those needed to activate PKC; insertion into the membrane may be a primary mechanism of PKC activation. PKC may be a long-term cell regulator which becomes inserted into the membrane upon appearance of the second messengers, calcium and diacylglycerol, and remains in an active membrane-bound state when the second messengers have been removed.

Mechanism of protein kinase C inhibition by sphingosine.

The in vitro mechanism by which sphingosine inhibits protein kinase C (PKC) was investigated by comparing enzyme activity and the physical associations of reaction components. Light scattering intensity measurements showed that sphingosine prevented the association of the substrate, histone, with micelles of Triton plus phosphatidylserine (PS). Addition of phosphatidylinositol (PI) or phosphatidylglycerol (PG) restored histone interaction. In direct correlation, both PI and PG were able to reverse inhibition of PKC activity by sphingosine. In Triton mixed micelles, neither PI nor PG alone would support PKC activity or substrate-lipid binding. Inhibition of PKC by positively charged sphingosine appeared to be related to simple charge neutralization of the lipid, thereby preventing interaction with PKC and/or its protein substrate.

Relative Quantification: Characterization of bias, variability and fold changes in mass spectrometry data from iTRAQ labeled peptides

Shotgun proteomics via mass spectrometry (MS) is a powerful technology for biomarker discovery that has the potential to lead to noninvasive disease screening mechanisms. Successful application of MS-based proteomics technologies for biomarker discovery requires accurate expectations of bias, reproducibility, variance, and the true detectable differences in platforms chosen for analyses. Characterization of the variability inherent in MS assays is vital and should affect interpretation of measurements of observed differences in biological samples. Here we describe observed biases, variance structure, and the ability to detect known differences in spike-in data sets for which true relative abundance among defined samples were known and were subsequently measured with the iTRAQ technology on two MS platforms. Global biases were observed within these data sets. Measured variability was a function of mean abundance. Fold changes were biased toward the null and variance of a fold change was a function of protein mass and abundance. The information presented herein will be valuable for experimental design and analysis of the resulting data.

Amino Acid-Directed Nucleic Acid Synthesis A Possible Mechamism in the Origin of Life

SummaryThe fact that proteins contain onlya-amino acids and that protein structure is determined by 3′ → 5′ linked ribonucleotides is postulated to be the result of the copolymerization of these molecules in the prebiotic environment. Ribonucleotides therefore represent partial degradation products and proteins represent a side reaction developing from copolymerization. The basic structural unit of copolymerization is a nucleotide substituted with an amino acid at the 2′ position. Characteristics of modern amino and ribonucleic acid structure are all consistent with and necessary for this hypothesis. The characteristics and individual base assignments of the code also provide strong support for origin from the postulated copolymers. All characteristics of the code can be accounted for by this single hypothesis.

Membrane association with multiple calcium ions: vitamin-K-dependent proteins, annexins and pentraxins

Peripheral membrane association with high calcium stoichiometry is shared by three families of proteins: annexins, pentraxins and vitamin-K-dependent proteins. Recent crystal structure determinations, biophysical studies of membrane binding and analyses of protein electrostatic properties offer striking and different concepts for membrane association by each of these protein families.

Myelin basic protein-enhanced fusion of membranes.

Myelin basic protein caused rapid aggregation of vesicles containing acidic phospholipids. Aggregation could be reversed by trypsin digestion of the myelin basic protein. Aggregated vesicles containing gel phase phospholipids or vesicles containing greater than 15 mol% lysolecithin underwent fusion. The extent of fusion was measured by irreversible changes in the light-scattering intensities or diffusion coefficients of the vesicles. Fusion was also measured by the fluorescence quenching which occurred when vesicles containing a covalently bound fluorophore. N-4-nitrobenzo-2-oxa-1,3-diazole, were fused with vesicles containing the covalently bound spin label, 4,4-dimethyl-oxazolidine-N-oxyl. The kinetics of fusion were first order in phospholipid and had half-times of 0.5-5 min depending on lysolecithin composition. This protein-enhanced membrane fusion may provide a valuable model system for studying some types of biological membrane fusions.

Enhancement of human protein C function by site-directed mutagenesis of the gamma-carboxyglutamic acid domain.

This study reports properties of site-directed mutants of human protein C that display enhanced calcium and/or membrane binding properties. Mutants containing the S11G modification all showed increased affinity for membranes at saturating calcium concentration. Ser-11 is unique to human protein C, whereas all other vitamin K-dependent proteins contain glycine. This site is located in a compact region of the protein, close to a suggested membrane contact site. Additional changes of H10Q or S12N resulted in proteins with lower calcium requirement for membrane contact but without further increase in membrane affinity at saturating calcium. Mutations Q32E and N33D did not, by themselves, alter membrane affinity to a significant degree. These mutations were included in other mutant proteins and may contribute somewhat to higher function in these mutants. This family of mutants helped discriminate events that are necessary for protein-membrane binding. These include calcium binding to the free protein and subsequent protein-membrane contact. Depending on conditions of the assay used, the mutants displayed increased activity of the corresponding activated protein C (APC) derivatives. The degree of enhanced activity (up to 10-fold) was dependent on the concentration of phospholipid and quality of phospholipid (± phosphatidylethanolamine) used in the assay. This was expected, because APC is active in its membrane-associated form, which can be regulated by changes in either the protein or phospholipid. As expected, the largest impact of the mutants occurred at low phospholipid concentration and in the absence of phosphatidylethanolamine. The anticoagulant activity of all proteins was stimulated by protein S, with the greatest impact on the enhanced mutants. Whereas plasma containing Factor V:R506Q was partially resistant to all forms of APC, the enhanced variants were more active than normal APC. Protein C variants with enhanced function present new reagents for study of coagulation and may offer improved materials for biomedical applications.

Temporal stability of the urinary proteome after kidney transplant: more sensitive than protein composition?

The temporal urinary proteome was examined in 4 groups of individuals in order to determine the temporal stability of diverse individuals with apparently good kidney health. The groups consisted of (1) healthy volunteers at zero time, 1 and 6 months, (2) kidney donors before and after surgery, (3) recipients immediately after surgery, and (4) successful kidney transplant recipients from 1 month to 4 years after transplant. Proteins were detected by reverse phase extraction of urine followed by MALDI-TOF profile and by iTRAQ analysis. Unusual components of the MALDI-TOF profiles found only in transplant subjects occurred at m/ z = 3370, 3441 and 3385 (human neutrophil defensins), 4303, 10350, and 11732 (beta-2 microglobulin, B2M). The peaks at m/ z = 4303 and 11732 were also quite intense among kidney donors following surgery. The peaks at m/ z = 4303 and 10350 in transplant recipients were associated with higher serum creatinine. Several additional proteins detected by iTRAQ were up-regulated in a manner that correlated closely with B2M. Overall, despite large differences between protein composition in different transplant recipients, there was remarkable stability for each individual as detected by either MALDI-TOF or iTRAQ analyses. These results suggested that, within limits, stability of profile components may be as important as protein content for definition of kidney health. Longitudinal study of urinary proteins from kidney recipients may demonstrate instability as a sensitive biomarker of adverse kidney health.