Michael Blaber

Proteinase-activated receptors, targets for kallikrein signaling

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR1 and PAR4, the endogenous enzymes responsible for activating PAR2 in settings other than the gastrointestinal system, where trypsin can activate PAR2, are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR1, PAR2, and PAR4; 2) PAR-dependent calcium signaling responses in cells expressing PAR1, PAR2, and PAR4 and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR4-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR1, PAR2, and PAR4 and to disarm/inhibit PAR1. Although hK5 and -6 were also able to activate PAR2, they failed to cause PAR4-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR2-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR1, PAR2, and PAR4.

Activation Profiles and Regulatory Cascades of the Human Kallikrein-Related Peptidases

The human kallikrein (KLK)-related peptidases are the largest family of serine peptidases, comprising 15 members (KLK1–15) and with the majority (KLK4–15) being identified only within the last decade. Members of this family are associated with important diseased states (including cancer, inflammation, and neurodegeneration) and have been utilized or proposed as clinically important biomarkers or therapeutic targets of interest. All human KLKs are synthesized as prepro-forms that are proteolytically processed to secreted pro-forms via the removal of an amino-terminal secretion signal peptide. The secreted inactive pro-KLKs are then activated extracellularly to mature peptidases by specific proteolytic release of their amino-terminal propeptide. Although a key step in the regulation of KLK function, details regarding the activation of the human pro-KLKs (i.e. the KLK “activome”) are unknown, to a significant extent, but have been postulated to involve “activation cascades” with other KLKs and endopeptidases. To characterize more completely the KLK activome, we have expressed from Escherichia coli individual KLK propeptides fused to the amino terminus of a soluble carrier protein. The ability of 12 different mature KLKs to process the 15 different pro-KLK peptide sequences has been determined. Various autolytic and cross-activation relationships identified using this system have subsequently been characterized using recombinant pro-KLK proteins. The results demonstrate the potential for extensive KLK activation cascades and, when combined with available data for the tissue-specific expression of the KLK family, permit the construction of specific regulatory cascades. One such tissue-specific cascade is proposed for the central nervous system.

Kallikrein-mediated cell signalling: targeting proteinase-activated receptors (PARs).

Abstract We tested the hypothesis that human tissue kallikreins (hKs) may regulate signal transduction by cleaving and activating proteinase-activated receptors (PARs). We found that hK5, 6 and 14 cleaved PAR N-terminal peptide sequences representing the cleavage/activation motifs of human PAR1 and PAR2 to yield receptor-activating peptides. hK5, 6 and 14 activated calcium signalling in rat PAR2-expressing (but not background) KNRK cells. Calcium signalling in HEK cells co-expressing human PAR1 and PAR2 was also triggered by hK14 (via PAR1 and PAR2) and hK6 (via PAR2). In isolated rat platelets that do not express PAR1, but signal via PAR4, hK14 also activated PAR-dependent calcium signalling responses and triggered aggregation. The aggregation response elicited by hK14 was in contrast to the lack of aggregation triggered by hK5 and 6. hK14 also caused vasorelaxation in a phenylephrine-preconstricted rat aorta ring assay and triggered oedema in an in vivo model of murine paw inflammation. We propose that, like thrombin and trypsin, the kallikreins must now be considered as important ‘hormonal’ regulators of tissue function, very likely acting in part via PARs.

Substrate Specificity of Human Kallikrein 6 SALT AND GLYCOSAMINOGLYCAN ACTIVATION EFFECTS

Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km = 38,667 s–1 mm–1). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Aβ amyloid peptide, reactive center loop of α1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1–4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.

Targeting kallikrein 6 proteolysis attenuates CNS inflammatory disease

Kallikrein 6 (K6, MSP) is a newly identified member of the Kallikrein family of serine proteases that is preferentially expressed in the adult central nervous system (CNS). We have previously demonstrated that K6 is abundantly expressed by inflammatory cells at sites of CNS inflammation and demyelination in animal models of multiple sclerosis (MS) and in human MS lesions. To test the hypothesis that this novel enzyme is a mediator of pathogenesis in CNS inflammatory disease, we have evaluated whether autonomously generated K6 antibodies alter the clinicopathological course of disease in murine proteolipid protein139‐151‐induced experimental autoimmune encephalomyelitis (PLP139‐151 EAE). We demonstrate that immunization of mice with recombinant K6 generates antibodies that block K6 enzymatic activity in vitro, including the breakdown of myelin basic protein (MBP), and that K6‐ immunized mice exhibit significantly delayed onset and severity of clinical deficits. Reduced clinical deficits were reflected in significantly less spinal cord pathology and meningeal inflammation and in reduced Th1 cellular responses in vivo and in vitro. These data demonstrate for the first time that K6 participates in enzymatic cascades mediating CNS inflammatory disease and that this unique enzyme may represent a novel therapeutic target for the treatment of progressive inflammatory disorders, including MS.

Structure and stability effects of mutations designed to increase the primary sequence symmetry within the core region of a beta-trefoil.

Human acidic fibroblast growth factor (FGF‐1) is a member of the β‐trefoil hyperfamily and exhibits a characteristic threefold symmetry of the tertiary structure. However, evidence of this symmetry is not readily apparent at the level of the primary sequence. This suggests that while selective pressures may exist to retain (or converge upon) a symmetric tertiary structure, other selective pressures have resulted in divergence of the primary sequence during evolution. Using intra‐chain and homologue sequence comparisons for 19 members of this family of proteins, we have designed mutants of FGF‐1 that constrain a subset of core‐packing residues to threefold symmetry at the level of the primary sequence. The consequences of these mutations regarding structure and stability were evaluated using a combination of X‐ray crystallography and differential scanning calorimetry. The mutational effects on structure and stability can be rationalized through the characterization of “microcavities” within the core detected using a 1.0Å probe radius. The results show that the symmetric constraint within the primary sequence is compatible with a well‐packed core and near wild‐type stability. However, despite the general maintenance of overall thermal stability, a noticeable increase in non‐two‐state denaturation follows the increase in primary sequence symmetry. Therefore, properties of folding, rather than stability, may contribute to the selective pressure for asymmetric primary core sequences within symmetric protein architectures.

Reversible Thermal Denaturation of Human FGF-1 Induced by Low Concentrations of Guanidine Hydrochloride

Human acidic fibroblast growth factor (FGF-1) is a powerful mitogen and angiogenic factor with an apparent melting temperature (Tm) in the physiological range. FGF-1 is an example of a protein that is regulated, in part, by stability-based mechanisms. For example, the low Tm of FGF-1 has been postulated to play an important role in the unusual endoplasmic reticulum-independent secretion of this growth factor. Despite the close relationship between function and stability, accurate thermodynamic parameters of unfolding for FGF-1 have been unavailable, presumably due to effects of irreversible thermal denaturation. Here we report the determination of thermodynamic parameters of unfolding (DeltaH, DeltaG, and DeltaCp) for FGF-1 using differential scanning calorimetry (DSC). The thermal denaturation is demonstrated to be two-state and reversible upon the addition of low concentrations of added guanidine hydrochloride (GuHCl). DeltaG values from the DSC studies are in excellent agreement with values from isothermal GuHCl denaturation monitored by fluorescence and circular dichroism (CD) spectroscopy. Furthermore, the results indicate that irreversible denaturation is closely associated with the formation of an unfolding intermediate. GuHCl appears to promote reversible two-state denaturation by initially preventing aggregation of this unfolding intermediate, and at subsequently higher concentrations, by preventing formation of the intermediate.

Kallikreins are associated with secondary progressive multiple sclerosis and promote neurodegeneration

Abstract Tissue kallikrein KLK1 and the kallikrein-related peptidases KLK2–15 are a subfamily of serine proteases that have defined or proposed roles in a range of central nervous system (CNS) and non-CNS pathologies. To further understand their potential activity in multiple sclerosis (MS), serum levels of KLK1, 6, 7, 8 and 10 were determined in 35 MS patients and 62 controls by quantitative fluorometric ELISA. Serum levels were then correlated with Expanded Disability Status Scale (EDSS) scores determined at the time of serological sampling or at last clinical follow-up. Serum levels of KLK1 and KLK6 were elevated in MS patients (p≤0.027), with highest levels associated with secondary progressive disease. Elevated KLK1 correlated with higher EDSS scores at the time of serum draw and KLK6 with future EDSS worsening in relapsing remitting patients (p≤0.007). Supporting the concept that KLK1 and KLK6 promote degenerative events associated with progressive MS, exposure of murine cortical neurons to either kallikrein promoted rapid neurite retraction and neuron loss. These novel findings suggest that KLK1 and KLK6 may serve as serological markers of progressive MS and contribute directly to the development of neurological disability by promoting axonal injury and neuron cell death.

1.70 A X-ray structure of human apo kallikrein 1: Structural changes upon peptide inhibitor/substrate binding

Human kallikreins are serine proteases that comprise a recently identified large and closely related 15‐member family. The kallikreins include both regulatory‐ and degradative‐type proteases, impacting a variety of physiological processes including regulation of blood pressure, neuronal health, and the inflammatory response. While the function of the majority of the kallikreins remains to be elucidated, two members are useful biomarkers for prostate cancer and several others are potentially useful biomarkers for breast cancer, Alzheimers, and Parkinsons disease. Human tissue kallikrein (human K1) is the best functionally characterized member of this family, and is known to play an important role in blood pressure regulation. As part of this function, human K1 exhibits unique dual‐substrate specificity in hydrolyzing low molecular weight kininogen between both Arg‐Ser and Met‐Lys sequences. We report the X‐ray crystal structure of mature, active recombinant human apo K1 at 1.70 Å resolution. The active site exhibits structural features intermediate between that of apo and pro forms of known kallikrein structures. The S2 to S2′ pockets demonstrate a variety of conformational changes in comparison to the porcine homolog of K1 in complex with peptide inhibitors, including the displacement of an extensive solvent network. These results indicate that the binding of a peptide substrate contributes to a structural rearrangement of the active‐site Ser 195 resulting in a catalytically competent juxtaposition with the active‐site His 57. The solvent networks within the S1 and S1′ pockets suggest how the Arg‐Ser and Met‐Lys dual substrate specificity of human K1 is accommodated. Proteins 2005.

Experimental support for the evolution of symmetric protein architecture from a simple peptide motif

The majority of protein architectures exhibit elements of structural symmetry, and “gene duplication and fusion” is the evolutionary mechanism generally hypothesized to be responsible for their emergence from simple peptide motifs. Despite the central importance of the gene duplication and fusion hypothesis, experimental support for a plausible evolutionary pathway for a specific protein architecture has yet to be effectively demonstrated. To address this question, a unique “top-down symmetric deconstruction” strategy was utilized to successfully identify a simple peptide motif capable of recapitulating, via gene duplication and fusion processes, a symmetric protein architecture (the threefold symmetric β-trefoil fold). The folding properties of intermediary forms in this deconstruction agree precisely with a previously proposed “conserved architecture” model for symmetric protein evolution. Furthermore, a route through foldable sequence-space between the simple peptide motif and extant protein fold is demonstrated. These results provide compelling experimental support for a plausible evolutionary pathway of symmetric protein architecture via gene duplication and fusion processes.