Hyesook Yoon

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.

A completed KLK activome profile: investigation of activation profiles of KLK9, 10, and 15

Abstract We previously reported the activation profiles of the human kallikrein-related peptidases (KLKs) as determined from a KLK pro-peptide fusion-protein system. That report described the activity profiles of 12 of the 15 mature KLKs versus the 15 different pro-KLK sequences. The missing profiles in the prior report, involving KLK9, 10, and 15, are now described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mass spectrometry, and N-terminal sequence analyses show that KLK9 and 10 exhibit low hydrolytic activities towards all of the 15 pro-KLK sequences, while KLK15 exhibits significant activity towards both Arg- and Lys-containing KLK pro-sequences. The ability of KLK15 to activate pro-KLK8, 12, and 14 is confirmed using recombinant pro-KLK proteins, and shown to be significant for activation of pro-KLK8 and 14, but not 12. These additional data for KLK9, 10, and 15 now permit a completed KLK activome profile, using a KLK pro-peptide fusion-protein system, to be described. The results suggest that KLK15, once activated, can potentially feed back into additional pro-KLK activation pathways. Conversely, KLK9 and 10, once activated, are unlikely to participate in further pro-KLK activation pathways, although similar to KLK1 they may activate other bioactive peptides.

Activation profiles of human kallikrein-related peptidases by proteases of the thrombostasis axis

The human kallikrein‐related peptidases (KLKs) comprise 15 members (KLK1–15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro‐forms (pro‐KLKs) that are activated extracellularly by specific proteolytic release of their N‐terminal pro‐peptide. This processing is a key step in the regulation of KLK function. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, we have characterized the ability of proteases associated with thrombostasis to hydrolyze the pro‐peptide sequences of the KLK family using a previously described pro‐KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro‐KLK proteins. Pro‐KLK6 and 14 can be activated by both plasmin and uPA, with plasmin being the best activator of pro‐KLK6 identified to date. Pro‐KLK11 and 12 can be activated by a broad‐spectrum of thrombostasis proteases, with thrombin exhibiting a high degree of selectivity for pro‐KLK12. The results show that proteases of the thrombostasis family can efficiently activate specific pro‐KLKs, demonstrating the potential for important regulatory interactions between these two major protease families.

Kallikrein 6 signals through PAR1 and PAR2 to promote neuron injury and exacerbate glutamate neurotoxicity

CNS trauma generates a proteolytic imbalance contributing to secondary injury, including axonopathy and neuron degeneration. Kallikrein 6 (Klk6) is a serine protease implicated in neurodegeneration, and here we investigate the role of protease‐activated receptors 1 (PAR1) and PAR2 in mediating these effects. First, we demonstrate Klk6 and the prototypical activator of PAR1, thrombin, as well as PAR1 and PAR2, are each elevated in murine experimental traumatic spinal cord injury (SCI) at acute or subacute time points. Recombinant Klk6 triggered extracellular signal‐regulated kinase (ERK1/2) signaling in cerebellar granule neurons and in the NSC34 spinal cord motoneuron cell line, in a phosphoinositide 3‐kinase and MEK‐dependent fashion. Importantly, lipopeptide inhibitors of PAR1 or PAR2, and PAR1 genetic deletion, each reduced Klk6‐ERK1/2 activation. In addition, Klk6 and thrombin promoted degeneration of cerebellar neurons and exacerbated glutamate neurotoxicity. Moreover, genetic deletion of PAR1 blocked thrombin‐mediated cerebellar neurotoxicity and reduced the neurotoxic effects of Klk6. Klk6 also increased glutamate‐mediated Bim signaling, poly‐ADP‐ribose polymerase cleavage and lactate dehydrogenase release in NSC34 motoneurons and these effects were blocked by PAR1 and PAR2 lipopeptide inhibitors. Taken together, these data point to a novel Klk6‐signaling axis in CNS neurons that is mediated by PAR1 and PAR2 and is positioned to contribute to neurodegeneration.

Functional Role of Kallikrein 6 in Regulating Immune Cell Survival

Background Kallikrein 6 (KLK6) is a newly identified member of the kallikrein family of secreted serine proteases that prior studies indicate is elevated at sites of central nervous system (CNS) inflammation and which shows regulated expression with T cell activation. Notably, KLK6 is also elevated in the serum of multiple sclerosis (MS) patients however its potential roles in immune function are unknown. Herein we specifically examine whether KLK6 alters immune cell survival and the possible mechanism by which this may occur. Methodology/Principal Findings Using murine whole splenocyte preparations and the human Jurkat T cell line we demonstrate that KLK6 robustly supports cell survival across a range of cell death paradigms. Recombinant KLK6 was shown to significantly reduce cell death under resting conditions and in response to camptothecin, dexamethasone, staurosporine and Fas-ligand. Moreover, KLK6-over expression in Jurkat T cells was shown to generate parallel pro-survival effects. In mixed splenocyte populations the vigorous immune cell survival promoting effects of KLK6 were shown to include both T and B lymphocytes, to occur with as little as 5 minutes of treatment, and to involve up regulation of the pro-survival protein B-cell lymphoma-extra large (Bcl-XL), and inhibition of the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim). The ability of KLK6 to promote survival of splenic T cells was also shown to be absent in cell preparations derived from PAR1 deficient mice. Conclusion/Significance KLK6 promotes lymphocyte survival by a mechanism that depends in part on activation of PAR1. These findings point to a novel molecular mechanism regulating lymphocyte survival that is likely to have relevance to a range of immunological responses that depend on apoptosis for immune clearance and maintenance of homeostasis.

Kallikrein 6 regulates early CNS demyelination in a viral model of multiple sclerosis

Kallikrein 6 (Klk6) is a secreted serine protease that is elevated in active multiple sclerosis lesions and patient sera. To further evaluate the involvement of Klk6 in chronic progressive demyelinating disease, we determined its expression in the brain and spinal cord of SJL mice infected with Theilers murine encephalomyelitis virus (TMEV) and assessed the effects of Klk6‐neutralizing antibodies on disease progression. Klk6 RNA expression was elevated in the brain and spinal cord by 7 days postinfection (dpi). Thereafter, Klk6 expression persisted primarily in the spinal cord reaching a peak of fivefold over controls at mid‐chronic stages (60 dpi–120 dpi). Significant elevations in Klk6 RNA were also induced in splenocytes stimulated with viral capsid proteins in vitro and in activated human acute monocytic leukemia cells. Klk6‐neutralizing antibodies reduced TMEV‐driven brain and spinal cord pathology and delayed‐type hypersensitivity (DTH) responses when examined at early chronic time points (40 dpi). Reductions in spinal cord pathology included a decrease in activated monocytes/microglia and reductions in the loss of myelin basic protein (MBP). By 180 dpi, pathology scores no longer differed between groups. These findings point to regulatory activities for Klk6 in the development and progression of central nervous system (CNS) inflammation and demyelination that can be effectively targeted through the early chronic stages with neutralizing antibody.

Critical role for PAR1 in kallikrein 6-mediated oligodendrogliopathy

Kallikrein 6 (KLK6) is a secreted serine protease preferentially expressed by oligodendroglia in CNS white matter. Elevated levels of KLK6 occur in actively demyelinating multiple sclerosis (MS) lesions and in cases of spinal cord injury (SCI), stroke, and glioblastoma. Taken with recent evidence establishing KLK6 as a CNS‐endogenous activator of protease‐activated receptors (PARs), we hypothesized that KLK6 activates a subset of PARs to regulate oligodendrocyte physiology and potentially pathophysiology. Here, primary oligodendrocyte cultures derived from wild type or PAR1‐deficient mice and the murine oligodendrocyte cell line, Oli‐neu, were used to demonstrate that Klk6 (rodent form) mediates loss of oligodendrocyte processes and impedes morphological differentiation of oligodendrocyte progenitor cells (OPCs) in a PAR1‐dependent fashion. Comparable gliopathy was also elicited by the canonical PAR1 agonist, thrombin, as well as PAR1‐activating peptides (PAR1‐APs). Klk6 also exacerbated ATP‐mediated oligodendrogliopathy in vitro, pointing to a potential role in augmenting excitotoxicity. In addition, Klk6 suppressed the expression of proteolipid protein (PLP) RNA in cultured oligodendrocytes by a mechanism involving PAR1‐mediated Erk1/2 signaling. Microinjection of PAR1 agonists, including Klk6 or PAR1‐APs, into the dorsal column white matter of PAR1+/+ but not PAR1−/− mice promoted vacuolating myelopathy and a loss of immunoreactivity for myelin basic protein (MBP) and CC‐1+ oligodendrocytes. These results demonstrate a functional role for Klk6‐PAR1 signaling in oligodendroglial pathophysiology and suggest that antagonists of PAR1 or its protease agonists may represent new modalities to moderate demyelination and to promote myelin regeneration in cases of CNS white matter injury or disease.

Activation Profiles of Human Kallikrein-related Peptidases by Matrix Metalloproteinases

Abstract The 15 human kallikrein-related peptidases (KLKs) are clinically important biomarkers and therapeutic targets of interest in inflammation, cancer, and neurodegenerative disease. KLKs are secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their amino-terminal pro-peptide, and this is a key step in their functional regulation. Physiologically relevant KLK regulatory cascades of activation have been described in skin desquamation and semen liquefaction, and work by a large number of investigators has elucidated pairwise and autolytic activation relationships among the KLKs with the potential for more extensive activation cascades. More recent work has asked whether functional intersection of KLKs with other types of regulatory proteases exists. Such studies show a capacity for members of the thrombostasis axis to act as broad activators of pro-KLKs. In the present report, we ask whether such functional intersection is possible between the KLKs and the members of the matrix metalloproteinase (MMP) family by evaluating the ability of the MMPs to activate pro-KLKs. The results identify MMP-20 as a broad activator of pro-KLKs, suggesting the potential for intersection of the KLK and MMP axes under pathological dysregulation of MMP-20 expression.

The thrombin receptor is a critical extracellular switch controlling myelination

Hemorrhagic white matter injuries in the perinatal period are a growing cause of cerebral palsy yet no neuroprotective strategies exist to prevent the devastating motor and cognitive deficits that ensue. We demonstrate that the thrombin receptor (protease‐activated receptor 1, PAR1) exhibits peak expression levels in the spinal cord at term and is a critical regulator of the myelination continuum from initiation to the final levels achieved. Specifically, PAR1 gene deletion resulted in earlier onset of spinal cord myelination, including substantially more Olig2‐positive oligodendrocytes, more myelinated axons, and higher proteolipid protein (PLP) levels at birth. In vitro, the highest levels of PAR1 were observed in oligodendrocyte progenitor cells (OPCs), being reduced with differentiation. In parallel, the expression of PLP and myelin basic protein (MBP), in addition to Olig2, were all significantly higher in cultures of PAR1−/− oligodendroglia. Moreover, application of a small molecule inhibitor of PAR1 (SCH79797) to OPCs in vitro increased PLP and MBP expression. Enhancements in myelination associated with PAR1 genetic deletion were also observed in adulthood as evidenced by higher amounts of MBP and thickened myelin sheaths across large, medium, and small diameter axons. Enriched spinal cord myelination in PAR1−/− mice was coupled to increases in extracellular‐signal‐regulated kinase 1/2 and AKT signaling developmentally. Nocturnal ambulation and rearing activity were also elevated in PAR1−/− mice. These studies identify the thrombin receptor as a powerful extracellular regulatory switch that could be readily targeted to improve myelin production in the face of white matter injury and disease. GLIA 2015;63:846–859

Kallikrein cascades in traumatic spinal cord injury: in vitro evidence for roles in axonopathy and neuron degeneration.

Kallikreins (KLKs) are a family of 15 secreted serine proteases with emerging roles in neurologic diseases. To illuminate their contributions to the pathophysiology of spinal cord injury (SCI), we evaluated acute through chronic changes in the immunohistochemical appearance of 6 KLKs (KLK1, KLK5, KLK6, KLK7, KLK8, and KLK9) in postmortem human traumatic SCI cases, quantified their RNA expression levels in experimental murine SCI, and assessed the impact of recombinant forms of each enzyme toward murine cortical neurons in vitro. Temporally and spatially distinct changes in KLK expression were observed with partially overlapping patterns between human and murine SCI, including peak elevations (or reductions) during the acute and subacute periods. Kallikrein 9 showed the most marked changes and remained chronically elevated. Importantly, a subset of KLKs (KLK1, KLK5, KLK6, KLK7, and KLK9) were neurotoxic toward primary neurons in vitro. Kallikrein immunoreactivity was also observed in association with swollen axons and retraction bulbs in the human SCI cases examined. Together, these findings demonstrate that elevated levels of a significant subset of KLKs are positioned to contribute to neurodegenerative changes in cases of CNS trauma and disease and, therefore, represent new potential targets for the development of neuroprotective strategies.