Investigating the role of glutathione and glutathione biosynthetic genes in the adaptation of Anopheles gambiae to insecticides

Malaria remains a serious public health challenge in the tropical world, with 584,000 deaths globally in 2013, of which 90% occurred in Africa, and mostly in pregnant women and children under the age of five. Anopheles gambiae (An. gambiae) is the principal malaria vector in Africa, where vector control measures involve the use of insecticides in the forms of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). The development of insecticides resistance mitigates these approaches. Glutathione (GSH) is widely distributed among all living organisms, and is associated with detoxification pathways, especially the Glutathione S-transferases (GSTs). Its direct involvement and relevance in insecticide resistance in An. gambiae has not been determined. Thus, this work examines the contribution of GSH, its biosynthetic genes (GCLM, GCLC) and their possible transcriptional regulator Nrf2 in insecticide resistance in An. gambiae sampled from agricultural setting (areas of intensive agriculture) and residential setting (domestic area). Bioinformatics analysis, W.H.O. adult susceptibility bioassays and molecular techniques were employed to investigate. Total RNA was first isolated from the adults An. gambiae mosquitoes raised from agricultural and residential field-caught larvae which had been either challenged or unchallenged with insecticides. Semi-quantitative RT-PCR using gel image densitometry was used to determine the expression levels of GCLM, GCLC genes and Nrf2. Bioinformatics’ results established the presence of putative AGAP010259 (AhR) and AGAP005300 (Nf2e1) transcription factor binding sites in An. gambiae GCLC and GCLM promoters in silico. An. gambiae s.l. studied here were highly resistant to DDT and permethrin but less resistant to bendiocarb. Both knockdown resistance (kdr) mutation variants L1014S and L1014F that confers resistance to pyrethroid insecticides were identified in both An. coluzzii and An. arabiensis sampled from northern Nigeria. The L1014F was much associated with An. coluzzii. A significant positive correlation (P=0.04) between the frequency of the L1014F point mutation and resistance to DDT and permethrin was observed. However, a weak or non-significant correlation (P=0.772) between the frequency of the L1014S point mutation and resistance was also found. L1014S and L1014F mutations co-occurred in both agricultural and residential settings with high frequencies. However, the frequencies of the two mutations were greater in the agricultural settings than in the residential settings. The levels of total, reduced and oxidized GSH were significantly higher in mosquitoes from agricultural sites than those from residential sites. Increased oxidized GSH levels appears to correlate with higher DDT resistance. The expression levels of GCLM, GCLC and Nrf2 were also significantly up-regulated in adults An. gambiae raised from agricultural and residential field-caught larvae when challenged with insecticide. However, there was higher constitutive expression of GCLM, GCLC and Nrf2 in mosquitoes from agricultural setting. The increased expression levels of these genes and also GSH levels in this population suggest their roles in the response and adaptation of An. gambiae to insecticide challenges. There exists the feasibility of using GSH status in An. gambiae to monitor adaptation and resistance to insecticides.

Redução dos níveis de Ocratoxina A por ação da enzima peroxidase

A ocratoxina A é um composto formado a partir do metabolismo secundário de fungos dos gêneros Aspergillus e Penicillium. Uma vez que a presença dessa micotoxina nos alimentos causa sérios danos à saúde humana e animal, surge o interesse pelo desenvolvimento de métodos que visem a redução dos seus níveis em diferentes matrizes. Diversos processos de descontaminação têm sido propostos, sendo que os métodos de redução biológica tem recebido destaque. Esses métodos consistem na aplicação de micro-organismos ou de suas enzimas, o que gera a biotransformação ou degradação da toxina produzindo metabólitos com menor ou nenhuma toxicidade. Diante disso, o objetivo geral do trabalho foi avaliar o efeito da peroxidase na redução dos níveis de ocratoxina A. As enzimas peroxidases testadas foram a comercial e a obtida do farelo de arroz. Para a extração enzimática foram utilizadas as frações granulométricas do farelo de arroz de 48 a 100 mesh, sendo estas frações caracterizadas quimicamente. A peroxidase foi extraída do farelo de arroz em tampão 10 mM pH 5,0 e purificada por partição trifásica, obtendo 77,1% de recuperação e 9,2 para o fator de purificação. O método utilizado para a extração da ocratoxina A do sistema aquoso foi por partição líquido-líquido utilizando como solvente o clorofórmio, sendo esse método validado segundo os parâmetros de linearidade (0,1 a 20 ng mL-1), coeficientes de correlação (0,9997) e de determinação (0,9994), e limites de detecção (0,02) e quantificação (0,03). A afinidade entre as peroxidases e a ocratoxina A foi verificada segundo os parâmetros de KM e Vmáx, resultando em 0,00027 mM e 0,000015 mM min-1, respectivamente, para a peroxidase comercial, e 0,0065 mM e 0,000031 mM min-1 para a obtida do farelo de arroz. Com relação aos percentuais de redução de ocratoxina A, foram avaliadas 3 proporções enzima:substrato (1:10, 1:5 e 8:1 para a comercial e 1:10, 1:5 e a com atividade de 0,063 U mL-1 para a do farelo), sendo que as proporções que forneceram maior redução foi a de 8:1 para a enzima comercial (0,063 U mL-1) e a correspondente a 0,063 U mL-1 para a enzima obtida do farelo. Os percentuais de redução de ocratoxina A foram de 59% para a peroxidase comercial em 300 min e 41% para a peroxidase do farelo de arroz em 1440 min. O efeito de adsorção da ocratoxina A pela enzima peroxidase foi descartado uma vez que foi realizada a sua hidrólise com a enzima pepsina e verificado um percentual de 2,7% de adsorção, demonstrando que a redução foi por ação enzimática. A enzima obtida de farelo de arroz com atividade de 0,063 U mL-1 foi aplicada em suco de uva tinto e branco. Observou-se que para o primeiro não houve redução significativa, enquanto que para o segundo a redução foi de 17%. Neste trabalho, então, foi possível verificar a capacidade de redução dos níveis da ocratoxina A pela enzima peroxidase, tanto em sistema aquoso como no suco de uva integral branco.

Interaction between mercury (Hg), arsenic (As) and selenium (Se) affects the activity of glutathione S-transferase in breast milk: possible relationship with fish and shellfish intake

Breast milk is regarded as an ideal source of nutrients for the growth and development of neonates, but it can also be a potential source of pollutants. Mothers can be exposed to different contaminants as a result of their lifestyle and environmental pollution. Mercury (Hg) and arsenic (As) could adversely affect the development of fetal and neonatal nervous system. Some fish and shellfish are rich in selenium (Se), an essential trace element that forms part of several enzymes related to the detoxification process, including glutathione S-transferase (GST). The goal of this study was to determine the interaction between Hg, As and Se and analyze its effect on the activity of GST in breast milk. Milk samples were collected from women between day 7 and 10 postpartum. The GST activity was determined spectrophotometrically; total Hg, As and Se concentrations were measured by atomic absorption spectrometry. To explain the possible association of Hg, As and Se concentrations with GST activity in breast milk, generalized linear models were constructed. The model explained 44% of the GST activity measured in breast milk. The GLM suggests that GST activity was positively correlated with Hg, As and Se concentrations. The activity of the enzyme was also explained by the frequency of consumption of marine fish and shellfish in the diet of the breastfeeding women.

High glucose mediates prooxidant and antioxidant enzyme activities in coronary endothelial cells

Objective: Excess levels of free radicals such as nitric oxide (NO) and superoxide anion (O2-)are associated with the pathogenesis of endothelial cell dysfunction in diabetes mellitus. This study was designed to investigate the underlying causes of oxidative stress in coronary microvascular endothelial cells (CMEC) exposed to hyperglycaemia. Methods: CMEC were cultured under normal (5.5 mmol/L) or high glucose (22 mmol/L)concentrations for 7 days. The activity and expression (protein level) of eNOS, iNOS, NAD(P)H oxidase and antioxidant enzymes, namely, superoxide dismutase (SOD), catalase and glutahione peroxidase (GPx) were investigated by specific activity assays and Western analyses,respectively while the effects of hyperglycaemia on nitrite and O2 - generation were investigated by Griess reaction and cytochrome C reduction assay, respectively. Results: Hyperglycaemia did not alter eNOS or iNOS protein expressions and overall nitrite generation, an index of NO production. However, it significantly reduced the levels of intracellular antioxidant glutathione by 50% (p<0.05) and increased the protein expressions and/or activities of p22-phox, a membrane-bound component of pro-oxidant NAD(P)H oxidase and antioxidant enzymes (p<0.05). Free radical-scavengers, namely, Tiron and MPG (0.1-1 mol/L) reduced hyperglycaemia-induced antioxidant enzyme activity and increased glutathione and nitrite generation to the levels observed in CMEC cultured in normoglycaemic medium (p<0.01). The differences in enzyme activity and expressions were independent of the increased osmolarity generated by high glucose levels as investigated by using equimolar concentrations of mannitol in parallel experiments. Conclusions: These results suggest that hyperglycaemia-induced oxidative stress may arise in CMEC as a result of enhanced prooxidant enzyme activity and diminished generation of 3 antioxidant glutathione. By increasing the antioxidant enzyme capacity CMEC may protect themselves against free radical-induced cell damage in diabetic conditions. The definitive version is available at http://www.blackwell-synergy.com

Effect of ambient levels of ozone on photosynthetic components and radical scavenging system in leaves of African cowpea varieties

Tropospheric ozone (O3), a main component of photochemical oxidants, adversely affects not only human health but also vegetation. To clarify the long-term effects of ambient levels of tropospheric ozone (O3) on photosynthetic components and radical scavenging system in the leaves of cowpea ( Vigna unguiculata L.), two African varieties, Blackeye and Asontem, were grown in open-top chambers and exposed to filtered air (FA), non-filtered air (NF) or non-filtered air with additional O3 of approximately 50 nl l-1. Ambient levels of O3 significantly reduced chlorophyll concentration, quantum yield and activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), thus contributing to the reduction in net photosynthetic rate at the reproductive growth stage of both varieties; with no significant variety difference in the sensitivity to O3. The O3-induced significant reduction in catalase activity was observed in Blackeye at vegetative and reproductive growth stages; and in Asontem at reproductive growth stage. On the other hand, exposure to O3 significantly increased ascorbate peroxidase activity in Blackeye at reproductive stage and did not significantly affect that in Blackeye at vegetative growth stage and that in Asontem at both growth stages. At reproductive growth stage, activities of monodehydroascorbate reductase and glutathione reductase were significantly increased by the exposure to O3 in both varieties. The results obtained in this study suggest that, although ascorbate peroxidase, monodehydroascorbate reductase and glutathione reductase played important roles in scavenging O3-induced reactive oxygen species in the leaves, radical scavenging ability of these enzymes is not sufficient to avoid detrimental effects of ambient levels of O3 on photosynthesis in both African cowpea varieties.

Seasonal variation of acetylcholinesterase, catalase, glutathione s transferase in different size of Pinctada radiata and metal pollution (Pb, Cd, Ni) in Persian Gulf

There are various tools for monitoring the concentration of pollutants on aquatic ecosystems. Today these studies are based on biological monitoring and biomarkers. The aim of this study was to measure the concentration of the acetylcholinesterase (AChE), glutathione S-transferase and catalase as biomarkers of heavy metal contamination in pearl oyster Pinctada radiata and their mechanism in aquatic ecosystems. Heavy metals lead, cadmium and nickel were measured in soft tissue and studied stations in four seasons. Samples were collected seasonally in Lavan stations, Hendurabi and Nakhilo (in the northern Persian Gulf) from spring 2013 to winter of that year by scuba diving. Pearl oysters are divided according to their shells size; shells separated from soft tissues and were transferred to the laboratory for analysis of heavy metals and enzymes. Moopam standard method for were used for measuring the concentration of heavy metals and for analyzing tissue concentrations of glutathione S-transferase in Clam the method recommended by Habig et al in 1974 were used. For measuring acetylcholinesterase Ellman method were used. Catalase contamination in pearl oyster in the supernatant obtained from the study based on the method homogeate soft tissue of mussels (Abei, 1974) was evaluated. The results showed that the concentration of lead has significant difference in sediments station, the concentration of lead in Lavan is significantly higher than the other two stations, This could be due to the movement of tanker, boats and floating refueling and with a considerable amount of wastewater containing oil and Petroleum into the water, and also due to precipitation and industrial discharges the lead in the region is increasing, land-disposed sewage sludge, has large concentrations of lead. Compare the results of this study with standards related and other similar studies at the regional and international level showed that pollutant concentration of heavy metals in all cases significantly less than all the standards and guide values associated. And also compared to other world research results have been far less than others, Being Less of the conclusion given in this research according that nickel is one of the indicators of oil pollution in the study area and emissions have been relatively low of oil. The concentration of acetylcholinesterase at several stations, in large and small sizes and in the seasons had no significant difference. Variations of catalase, and glutathione S-transferase were almost similar to each other and parameters, station and seasons were significantly different in the concentrations of these enzymes. The effects and interaction between various parameters indicate that following parameters has impact on the concentration of catalase and glutathione S-transferase. Stations; Seasonal changes in antioxidant enzymes related to (assuming a constant in salinity and oxygen) to age, reproductive cycle, availability of food and water temperature. With increasing temperature at warm season, antioxidant enzymes were increase, with increasing temperature and abundance of food in the environment the amount of antioxidant enzymes may increase. The presence of the enzyme concentration may indicate that the higher levels of the enzyme to eliminate ROS activities to be any healthier situation. At the time of gonads maturation and spawning season catalase activity increases. This study also indicates that catalase was significantly higher in the warm season. Due to low pollutants of heavy metals in the study area, a lower level of contaminants were observed in shellfish tissue incidents of international standards and strong correlation between the amount of heavy metal contamination in pearl oyster tissue and enzymes was not observed. Therefore, we can say that the pearl oyster remains in a healthy condition and the amount of enzyme is normal.

Aplicação de peroxidase para degradação de deoxinivalenol

Deoxinivalenol (DON), uma das principais micotoxinas encontradas em matrizes alimentares, é um composto químico que possui em sua estrutura um anel epóxido que lhe confere alto grau de toxicidade. A aplicação de enzimas em processos de degradação de DON vem se destacando, pela estabilidade durante o processo reacional e baixo custo de produção. O objetivo desse trabalho foi estudar o potencial de peroxidase proveniente de farelo de arroz (FA) e farelo de soja (FS) para degradar DON. As condições de obtenção da PO a partir de FA foram definidas por planejamento experimental DCCR 23 , sendo extraída de 5 g de farelo com 50 mL de tampão fosfato 0,04 mol L-1 pH 5, agitados orbitalmente durante 60 min a 100 rpm, e para a PO obtida de FS as condições diferenciaram somente quanto a solução extratora, tampão fosfato 0,01 mol L-1 pH 4,7. A técnica que apresentou melhores índices de purificação para a enzima foi a partição trifásica apresentando fator de purificação e recuperação de 5,6 e 50 % para a obtida de FA e 13,61 e 50 % para FS. A PO de FA apresentou maior atividade em tampão fosfato 5 mmol L-1 pH 5,5 para as formas bruta e pura, diferindo na temperatura de reação de 25 °C e 10 °C, KM de 0,15 e 0,06 mmol L-1 e Vmáx de 769 e 667 U mg-1 , respectivamente. A PO de FS as condições foram: tampão fosfato 5 mmol L-1 pH 5, reação a 35 e 30 °C durante 10 e 5 min, KM de 0,17 e 0,05 mmol L-1 e Vmáx de 196 e 182 U mg-1 , respectivamente. A PO de FA demonstrou maior estabilidade em pH 5 enquanto que a de FS em pH 6, ambas enzimas apresentaram maior estabilidade térmica a 0 °C, as massas moleculares encontradas por eletroforese foram 41 e 34 kDa, respectivamente. Ao final das etapas de obtenção, purificação e caracterização obteve-se uma atividade específica de 116 e 794 U.mg-1 , e 4363 e 17453 U g-1 , respectivamente para PO de FA e FS. A determinação de DON e De-DON foi realizada por HPLC-DAD e LC-ESI-MS/MS para avaliação dos ensaios de degradação. A enzima comercial HRP, mostrou maior potencial de redução sobre DON (55% após 1 h de reação), no entanto em 3 h de reação, a concentração inicial da micotoxina DON foi verificada, o que evidencia que a redução pode ocorrer por adsorção ou por formação de um composto de degradação que apresente a mesma massa molecular. O emprego da enzima PO obtida de FA e FS na degradação necessita de uma avaliação cinética micotoxicologica para definição das condições de redução significativa dos níveis de DON.

Glutathione Transferases as Detoxification Agents: Structural and Functional Studies

Glutathione transferases (GSTs) are a diverse family of enzymes that catalyze the glutathione-dependent detoxification of toxic compounds. GSTs are responsible for the conjugation of the tripeptide glutathione (GSH) to a wide range of electrophilic substrates. These include industrial pollutants, drugs, genotoxic carcinogen metabolites, antibiotics, insecticides and herbicides. In light of applications in biomedicine and biotechnology as cellular detoxification agents, detailed structural and functional studies of GSTs are required. Plant tau class GSTs play crucial catalytic and non-catalytic roles in cellular xenobiotic detoxification process in agronomically important crops. The abundant existence of GSTs in Glycine max and their ability to provide resistance to abiotic and biotic stresses such as herbicide tolerance is of great interest in agriculture because they provide effective and suitable tools for selective weed control. Structural and catalytic studies on tau class GST isoenzymes from Glycine max (GmGSTU10-10, GmGSTU chimeric clone 14 (Sh14), and GmGSTU2-2) were performed. Crystal structures of GmGSTU10-10 in complex with glutathione sulfenic acid (GSOH) and Sh14 in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH) were determined by molecular replacement at 1.6 Å and 1.75 Å, respectively. Major structural variations that affect substrate recognition and catalytic mechanism were revealed in the upper part of helix H4 and helix H9 of GmGSTU10-10. Structural analysis of Sh14 showed that the Trp114Cys point mutation is responsible for the enhanced catalytic activity of the enzyme. Furthermore, two salt bridges that trigger an allosteric effect between the H-sites were identified at the dimer interface between Glu66 and Lys104. The 3D structure of GmGSTU2-2 was predicted using homology modeling. Structural and phylogenetic analysis suggested GmGSTU2-2 shares residues that are crucial for the catalytic activity of other tau class GSTs–Phe10, Trp11, Ser13, Arg20, Tyr30, Leu37, Lys40, Lys53, Ile54, Glu66 and Ser67. This indicates that the catalytic and ligand binding site in GmGSTU2-2 are well-conserved. Nevertheless, at the ligandin binding site a significant variation was observed. Tyr32 is replaced by Ser32 in GmGSTU2-2 and thismay affect the ligand recognition and binding properties of GmGSTU2-2. Moreover, docking studies revealed important amino acid residues in the hydrophobic binding site that can affect the substrate specificity of the enzyme. Phe10, Pro12, Phe15, Leu37, Phe107, Trp114, Trp163, Phe208, Ile212, and Phe216 could form the hydrophobic ligand binding site and bind fluorodifen. Additionally, side chains of Arg111 and Lys215 could stabilize the binding through hydrogen bonds with the –NO2 groups of fluorodifen. GST gene family from the pathogenic soil bacterium Agrobacterium tumefaciens C58 was characterized and eight GST-like proteins in A. tumefaciens (AtuGSTs) were identified. Phylogenetic analysis revealed that four members of AtuGSTs belong to a previously recognized bacterial beta GST class and one member to theta class. Nevertheless, three AtuGSTs do not belong to any previously known GST classes. The 3D structures of AtuGSTs were predicted using homology modeling. Comparative structural and sequence analysis of the AtuGSTs showed local sequence and structural characteristics between different GST isoenzymes and classes. Interactions at the G-site are conserved, however, significant variations were seen at the active site and the H5b helix at the C-terminal domain. H5b contributes to the formation of the hydrophobic ligand binding site and is responsible for recognition of the electrophilic moiety of the xenobiotic. It is noted that the position of H5b varies among models, thus providing different specificities. Moreover, AtuGSTs appear to form functional dimers through diverse modes. AtuGST1, AtuGST3, AtuGST4 and AtuGST8 use hydrophobic ‘lock–and–key’-like motifs whereas the dimer interface of AtuGST2, AtuGST5, AtuGST6 and AtuGST7 is dominated by polar interactions. These results suggested that AtuGSTs could be involved in a broad range of biological functions including stress tolerance and detoxification of toxic compounds.

Characterization of glutathione S transferases from the plant-parasitic nematode, Bursaphelenchus xylophilus

We have previously identified two secreted glutathione S-transferases (GST) expressed in the pharyngeal gland cell of Bursaphelenchus xylophilus, which are upregulated post infection of the host. This study examines the functional role of GSTs in B. xylophilus biology. We analysed the expression profiles of all predicted GSTs in the genome and the results showed that they belong to kappa and cytosolic subfamilies and the majority are upregulated post infection of the host. A small percentage is potentially secreted and none is downregulated post infection of the host. One secreted protein was confirmed as a functional GST and is within a cluster that showed the highest expression fold change in infection. This enzyme has a protective activity that may involve host defences, namely in the presence of terpenoid compounds and peroxide products. These results suggest that GSTs secreted into the host participate in the detoxification of host-derived defence compounds and enable successful parasitism.

Glutathione and abscisic acid supplementation influences somatic embryo maturation and hormone endogenous levels during somatic embryogenesis in Podocarpus lambertii Klotzsch ex Endl.

Here we propose a protocol for embryogenic cultures induction, proliferation and maturation for the Brazilian conifer Podocarpus lambertii, and investigated the effect of abscisic acid (ABA) and glutathione (GSH) supplementation on the maturation phase. ABA, zeatin (Z) and salicylic acid (SA) endogenous levels were quantified. Number of somatic embryos obtained in ABA-supplemented treatment was signifi- cant higher than in ABA-free treatment, showing the relevance of ABA supplementation during somatic embryos maturation. Histological analysis showed the stereotyped sequence of developmental stages in conifer somatic embryos, reaching the late torpedo-staged embryo. GSH supplementation in maturation culture medium improved the somatic embryos number and morphological features. GSH 0 mM and GSH 0.1 mM treatments correlated with a decreased ABA endogenous level during maturation, while GSH 0.5 mM treatment showed constantlevels. Alltreatments resulted in decreased Z endogenous levels, supporting the concept that cytokinins are important during the initial cell division but not for the later stages of embryo development. The lowest SA levels found in GSH 0.5 mM treatment were coincident with early embryonic development, and this treatment resulted in the highest development of somatic embryos. Thus, a correlation between lower SA levels and improved somatic embryo formation can be hypothesized

Ethyl-eicosapentaenoic acid in first-episode psychosis : a 1H-MRS study

Ethyl-eicosapentaenoic acid (E-EPA) is an omega-3 fatty acid that has been used in a range of neuropsychiatric conditions with some benefits. However, its mechanism of action is unknown. Here, we investigate its effects on in vivo brain metabolism in first-episode psychosis (FEP). Proton magnetic resonance spectroscopy at 3 T was performed in the temporal lobes of 24 FEP patients before and after 12 weeks of treatment in the context of a larger double-blind, placebo-controlled E-EPA augmentation study. Treatment group effects for glutathione (F1,12=6.1, p=0.03), and a hemisphere-by-group interaction for glutamine/glutamate (F1,20=4.4, p=0.049) were found. Glutathione increased bilaterally and glutamate/glutamine increased in the left hemisphere following E-EPA administration. Improvement in negative symptoms correlated with metabolic brain changes, particularly glutathione (r=-0.57). These results suggest that E-EPA augmentation alters glutathione availability and modulates the glutamine/glutamate cycle in early psychosis, with some of the metabolic brain changes being correlated with negative symptom improvement. Larger confirmatory studies of these postulated metabolic brain effects of E-EPA are warranted.

Selective cleavage of human sex hormone-binding globulin by kallikrein-related peptidases and effects on androgen action in LNCaP prostate cancer cells

Stimulation of the androgen receptor via bioavailable androgens, including testosterone and testosterone metabolites, is a key driver of prostate development and the early stages of prostate cancer. Androgens are hydrophobic and as such require carrier proteins, including sex hormone-binding globulin (SHBG), to enable efficient distribution from sites of biosynthesis to target tissues. The similarly hydrophobic corticosteroids also require a carrier protein whose affinity for steroid is modulated by proteolysis. However, proteolytic mechanisms regulating the SHBG/androgen complex have not been reported. Here, we show that the cancer-associated serine proteases, kallikrein-related peptidase (KLK)4 and KLK14, bind strongly to SHBG in glutathione S-transferase interaction analyses. Further, we demonstrate that active KLK4 and KLK14 cleave human SHBG at unique sites and in an androgen-dependent manner. KLK4 separated androgen-free SHBG into its two laminin G-like (LG) domains that were subsequently proteolytically stable even after prolonged digestion, whereas a catalytically equivalent amount of KLK14 reduced SHBG to small peptide fragments over the same period. Conversely, proteolysis of 5α-dihydrotestosterone (DHT)-bound SHBG was similar for both KLKs and left the steroid binding LG4 domain intact. Characterization of this proteolysis fragment by [(3)H]-labeled DHT binding assays revealed that it retained identical affinity for androgen compared with full-length SHBG (dissociation constant = 1.92 nM). Consistent with this, both full-length SHBG and SHBG-LG4 significantly increased DHT-mediated transcriptional activity of the androgen receptor compared with DHT delivered without carrier protein. Collectively, these data provide the first evidence that SHBG is a target for proteolysis and demonstrate that a stable fragment derived from proteolysis of steroid-bound SHBG retains binding function in vitro.