Proteomic analysis of human cervico-vaginal fluid

Human cervico-vaginal fluid (CVF) is a mixture of fluids originating from the vagina, cervix, endometrium, and oviduct. CVF has been shown to play an important role in protecting the vagina from infection. We used "bottom-up" proteomic approaches to characterize the protein repertoire of human CVF. We applied two different sample prefractionation methods, one-dimensional-SDS-PAGE (1D-SDS-PAGE) and strong cation-exchange chromatography, followed by LC-MS/MS and bioinformatic analysis. We identified a total of 685 proteins. Strong cation-exchange chromatography prefractionation resulted in a larger number of proteins identified when compared with 1D-SDS-PAGE. Extracellular or membrane proteins made up 30% of the proteins identified, according to Genome Ontology (GO) classifications. We confirmed the presence of defense-related proteins, such as haptoglobin, defensins, and lactoferrin; and identified new ones such as azurocidin and dermcidin. We also identified many serine and cysteine proteases, including 6 members of the kallikrein family (KLKs 6, 7, 10, 11, 12, and 13). The same KLKs were also confirmed quantitatively by ELISA assays. Knowledge of the CVF proteome will aid in the discovery of potential biomarkers for gynecological malignancies and infections and provide additional clues for its physiological functions.     

Chemical genetics reveals a complex functional ground state of neural stem cells

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain holds promise for the treatment of neurological diseases and has yielded new insight into brain cancer. However, the complete repertoire of signaling pathways that governs the proliferation and self-renewal of NSCs, which we refer to as the 'ground state', remains largely uncharacterized. Although the candidate gene approach has uncovered vital pathways in NSC biology, so far only a few highly studied pathways have been investigated. Based on the intimate relationship between NSC self-renewal and neurosphere proliferation, we undertook a chemical genetic screen for inhibitors of neurosphere proliferation in order to probe the operational circuitry of the NSC. The screen recovered small molecules known to affect neurotransmission pathways previously thought to operate primarily in the mature central nervous system; these compounds also had potent inhibitory effects on cultures enriched for brain cancer stem cells. These results suggest that clinically approved neuromodulators may remodel the mature central nervous system and find application in the treatment of brain cancer.     

A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation

Desquamation of the stratum corneum is a serine protease-dependent process. Two members of the human tissue kallikrein (KLK) family of (chymo)tryptic-like serine proteases, KLK5 and KLK7, are implicated in desquamation by digestion of (corneo)desmosomes and inhibition by desquamation-related serine protease inhibitors (SPIs). However, the epidermal localization and specificity of additional KLKs also supports a role for these enzymes in desquamation. This study aims to delineate the probable contribution of KLK1, KLK5, KLK6, KLK13, and KLK14 to desquamation by examining their interactions, in vitro, with: 1) colocalized SPI, lympho-epithelial Kazal-type-related inhibitor (LEKTI, four recombinant fragments containing inhibitory domains 1-6 (rLEKTI(1-6)), domains 6-8 and partial domain 9 (rLEKTI(6-9')), domains 9-12 (rLEKTI(9-12)), and domains 12-15 (rLEKTI(12-15)), secretory leukocyte protease inhibitor, and elafin and 2) their ability to digest the (corneo)desmosomal cadherin, desmoglein 1. KLK1 was not inhibited by any SPI tested. KLK5, KLK6, KLK13, and KLK14 were potently inhibited by rLEKTI(1-6), rLEKTI(6-9'), and rLEKTI(9-12) with Ki values in the range of 2.3-28.4 nm, 6.1-221 nm, and 2.7-416 nm for each respective fragment. Only KLK5 was inhibited by rLEKTI(12-15) (Ki = 21.8 nm). No KLK was inhibited by secretory leukocyte protease inhibitor or elafin. Apart from KLK13, all KLKs digested the ectodomain of desmoglein 1 within cadherin repeats, Ca2+ binding sites, or in the juxtamembrane region. Our study indicates that multiple KLKs may participate in desquamation through cleavage of desmoglein 1 and regulation by LEKTI. These findings may have clinical implications for the treatment of skin disorders in which KLK activity is elevated.     

Functional Roles of Human Kallikrein-related Peptidases

Kallikrein-related peptidases constitute a single family of 15 (chymo)trypsin-like proteases (KLK1–15) with pleiotropic physiological roles. Aberrant regulation of KLKs has been associated with diverse diseases such as hypertension, renal dysfunction, skin disorders, inflammation, neurodegeneration, and cancer. Recent studies suggested that coordinated activation and regulation of KLK activity are achieved via a complex network of interactions referred to as the “KLK activome.” However, it remains to be validated whether these hypothetical KLK activation cascade pathways are operative in vivo. In addition, KLKs have emerged as versatile signaling molecules. In summary, KLKs represent attractive biomarkers for clinical applications and potential therapeutic targets for common human pathologies.     

Effects of long-term androgen administration on breast tissue of female-to-male transsexuals.

Our aim was to examine the effects of androgen administration on breast tissue histology of female-to-male transsexuals and to study the immunohistochemical expression of three human tissue kallikreins, hK3 (PSA), hK6, and hK10. We studied 23 female-to-male transsexuals who were treated with injectable testosterone for 18-24 months. We also used 10 control female breast tissues. All tissues were fixed in buffered formalin, embedded in paraffin, and examined by hematoxylin-eosin staining and immunohistochemical staining for PSA, hK6, and hK10. Females treated with androgens exhibited similar involutionary changes as those seen in breast of menopausal women, such as marked reduction of glandular tissue, involution of the lobuloalveolar structures, and prominence of fibrous connective tissue, but presence of only small amounts of fat tissue. Fibrocystic lesions were generally not observed. In immunohistochemistry, in control breast tissues, we found moderate to strong cytoplasmic immunoexpression of hK6 and hK10 in the epithelial ductal and lobuloalveolar structures, but myoepithelial cells were negative. Luminal secretions were also positive. In menopausal breast, the immunoexpression of hK6 and hK10 was weaker and focal. No control case showed immunoexpression for PSA. In female-to-male transsexuals, one case showed focal PSA cytoplasmic immunoexpression in the epithelium of moderately involuting lobules. Long-term administration of androgens in female-to-male transsexuals causes marked reduction of glandular tissue and prominence of fibrous connective tissue. These changes are similar to those observed at the end-stage of menopausal mammary involution.     

KLK12 Is a Novel Serine Protease and a New Member of the Human Kallikrein Gene Family—Differential Expression in Breast Cancer

Kallikreins are a subgroup of serine proteases that are involved in the posttranslational processing of polypeptide precursors. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. In rodents, kallikreins are encoded by a large multigene family, but in humans, only three genes have been identified. By using the positional candidate approach, we were able to identify a new kallikrein-like gene, tentatively named KLK12 (for kallikrein gene 12). This new gene maps to chromosome 19q13.3–q13.4, is formed of five coding exons, and shows structural similarity to serine proteases and other known kallikreins. KLK12 is expressed in a variety of tissues including salivary gland, stomach, uterus, lung, thymus, prostate, colon, brain, breast, thyroid, and trachea. We identified three splicing forms of KLK12 that are expressed in many tissues. Our preliminary results indicate that the expression of KLK12 is down-regulated at the mRNA level in breast cancer tissues and is up-regulated by steroid hormones in breast and prostate cancer cell lines. This gene may be involved in the pathogenesis and/or progression of certain cancer types and may find applicability as a novel cancer biomarker.