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911.
Jayita Guhaniyogi Istvan Sohar Kalyan Das Ann M. Stock Peter Lobel 《The Journal of biological chemistry》2009,284(6):3985-3997
Late infantile neuronal ceroid lipofuscinosis is a fatal childhood
neurological disorder caused by a deficiency in the lysosomal protease
tripeptidyl-peptidase 1 (TPP1). TPP1 represents the only known mammalian
member of the S53 family of serine proteases, a group characterized by a
subtilisin-like fold, a Ser-Glu-Asp catalytic triad, and an acidic pH optimum.
TPP1 is synthesized as an inactive proenzyme (pro-TPP1) that is
proteolytically processed into the active enzyme after exposure to low pH
in vitro or targeting to the lysosome in vivo. In this
study, we describe an endoglycosidase H-deglycosylated form of TPP1 containing
four Asn-linked N-acetylglucosamines that is indistinguishable from
fully glycosylated TPP1 in terms of autocatalytic processing of the proform
and enzymatic properties of the mature protease. The crystal structure of
deglycosylated pro-TPP1 was determined at 1.85 Å resolution. A large
151-residue C-shaped prodomain makes extensive contacts as it wraps around the
surface of the catalytic domain with the two domains connected by a 24-residue
flexible linker that passes through the substrate-binding groove. The
proenzyme structure reveals suboptimal catalytic triad geometry with its
propiece linker partially blocking the substrate-binding site, which together
serve to prevent premature activation of the protease. Finally, we have
identified numerous processing intermediates and propose a structural model
that explains the pathway for TPP1 activation in vitro. These data
provide new insights into TPP1 function and represent a valuable resource for
constructing improved TPP1 variants for treatment of late infantile neuronal
ceroid lipofuscinosis.Late infantile neuronal ceroid lipofuscinosis
(LINCL)3 (OMIM number
204500) is a neurodegenerative lysosomal storage disease of childhood that
presents typically between the ages of 2 and 4 years with the onset of
seizures. Disease progression is reflected by blindness, dementia, mental
retardation, and an increase in the severity of seizures. LINCL is always
fatal, and the life span of patients is typically 6-15 years. LINCL is caused
by mutations in TPP1 (previously named CLN2, for ceroid
lipofuscinosis neuronal type 2 gene)
(1), which normally encodes a
lysosomal protease, tripeptidyl-peptidase 1 (TPP1, EC 3.4.14.9)
(2,
3).There is currently no treatment of demonstrated efficacy for LINCL, but
promising progress is being made in some directions. Proof-of-principle for
virus-mediated gene therapy has been established in a mouse model of LINCL,
with a significant improvement in disease phenotype achieved with the use of
adeno-associated virus vectors expressing TPP1
(4-7).
Affected children have also been treated with adeno-associated virus vectors,
although it is too soon to determine whether significant clinical benefits
have been achieved in these early trials
(8). Enzyme replacement
therapy, an approach that has proven successful in a number of other lysosomal
storage diseases, has also been investigated in LINCL. Purified recombinant
human TPP1 that contains the mannose 6-phosphate lysosomal targeting
modification can be taken up by LINCL fibroblasts where it degrades storage
material (9), and the protein
has been introduced into the cerebrospinal fluid of the LINCL mouse model via
intraventricular injection, resulting in significant uptake into the brain and
some correction of neuropathology
(10).For therapeutic approaches that rely upon replacing a mutant gene product
with a functional protein via recombinant methods, e.g. gene and
enzyme replacement therapy, a thorough understanding of the biological and
biophysical properties of the protein in question are essential for success.
Thus, for LINCL, considerable effort has been directed toward the
investigation of TPP1, and as a result, this is a well characterized enzyme at
the functional and molecular levels (reviewed in Refs.
11,
12). TPP1 encodes a
563-residue preproprotein with a cleavable N-terminal 19-residue signal
sequence. The proenzyme (residues 20-563) is a soluble monomer that undergoes
proteolytic cleavage in the lysosome, converting the zymogen to an active,
mature protease (residues 196-563)
(1). Studies on purified
pro-TPP1 demonstrate that maturation is autocatalytic in vitro
(13,
14) but may involve other
proteases in vivo
(15). TPP1 is glycosylated,
and its N-linked oligosaccharides have been implicated in maturation,
activity, targeting, and stability of the processed enzyme
(16,
17).TPP1 is a serine protease
(14) that possesses two
catalytic functions as follows: a primary tripeptidyl exopeptidase activity
with a pH optimum of ∼5.0 that catalyzes the sequential release of
tripeptides from the unsubstituted N termini of substrates
(18), and a much weaker
endoproteolytic activity with a pH optimum of ∼3.0
(19). TPP1 exhibits broad
substrate specificity (20) and
is the only mammalian member of the S53 sedolisin family (reviewed in Ref.
21), which includes a number
of unusual bacterial serine peptidases
(22). High resolution crystal
structures of both free and inhibitor-bound complexes have been determined for
three bacterial members of this family (sedolisin
(23-26),
kumamolisin (27,
28), and kumamolisin-As
(29,
30)), and for one
(kumamolisin), the structure of a mutant, inactive precursor form has also
been obtained (28). These
proteins share a common subtilisin-like fold, an octahedrally coordinated
calcium-binding site, and an active site that contains an unusual Ser-Glu-Asp
(SED) catalytic triad, rather than the Ser-His-Asp (SHD) triad of subtilisin
(31,
32). Chemical modification
studies of TPP1 have revealed that Ser475 is the active site
nucleophile (14). Modeling
studies suggest that Glu272 and Asp276 complete the
catalytic triad and that Asp360 is homologous to the conserved Asn
in the subtilisin family in its role in stabilization of the oxyanion of the
tetrahedral intermediate during catalysis
(33). Site-directed
mutagenesis studies are consistent with these conclusions
(14,
34).A detailed understanding of the tertiary structure of TPP1 may have
implications for developing or improving therapeutic strategies. First, a high
resolution model would provide the basis for targeted protein engineering
efforts to design TPP1 derivatives with increased half-life prior to and/or
upon delivery to the lysosome. Successful creation of a longer lived TPP1
molecule could significantly enhance gene or enzyme replacement approaches to
LINCL. Second, a structural model for TPP1 could be valuable in designing
derivatives tagged with protein transduction domains to facilitate crossing of
the blood-brain barrier for delivery to the central nervous system from the
bloodstream. In this study, we present the crystal structure of the proform of
human TPP1 at 1.85 Å resolution. This model provides novel insights into
the structural basis for the pH-induced auto-activation of the proform of
TPP1. A structure of glycosylated pro-TPP1 has been independently determined,
displaying features similar to those of deglycosylated
TPP1.4 相似文献
912.
913.
Derek C. Cole Joseph R. Stock Anthony F. Kreft Madelene Antane Suzan H. Aschmies Kevin P. Atchison David S. Casebier Thomas A. Comery George Diamantidis John W. Ellingboe Boyd L. Harrison Yun Hu Mei Jin Dennis M. Kubrak Peimin Lu Charles W. Mann Robert L. Martone William J. Moore Aram Oganesian David R. Riddell J. Steven Jacobsen 《Bioorganic & medicinal chemistry letters》2009,19(3):926-929
Accumulation of beta-amyloid (Aβ), produced by the proteolytic cleavage of amyloid precursor protein (APP) by β- and γ-secretase, is widely believed to be associated with Alzheimer’s disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) γ-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-β,β-diethylalaninol 7.b.2 (Aβ 40/42 EC50 = 28 nM), which is efficacious in reduction of Aβ production in vivo. 相似文献
914.
Mobility patterns affect the loads placed on the lower limbs during locomotion and may influence variation in lower limb diaphyseal robusticity and shape. This relationship commonly forms the basis for inferring mobility patterns from hominin fossil and skeletal remains. This study assesses the correspondence between athletic histories, varying by loading intensity, repetition and directionality, measured using a recall questionnaire, and peripheral quantitative computed tomography‐derived measurements of tibial diaphysis rigidity and shape. Participants included male university varsity cross‐country runners (n = 15), field hockey players (n = 15), and controls (n = 20) [mean age: 22.1 (SD +/? 2.6) years]. Measurements of tibial rigidity (including J, %CA, Imax, Imin, and average cortical thickness) of both runners and field hockey players were greater than controls (P ≤ 0.05). Differences in tibial shape (Imax/Imin, P ≤ 0.05) between runners and hockey players reflect pronounced maximum plane (Imax) rigidity in runners, and more symmetrical hypertrophy (Imax, Imin) among hockey players. This corresponds with the generally unidirectional locomotor patterns of runners, and the multidirectional patterns of hockey players. These results support the relationship between mobility and tibial diaphysis morphology as it is generally interpreted in the anthropological literature, with greater levels of mobility associated with increased diaphyseal robusticity and shape variation. Although exercise intensity may be the primary influence on these properties, the repetitiveness of the activity also deserves consideration. In conclusion, bone morphological patterns can reflect habitual behaviors, with adaptation to locomotor activities likely contributing to variation in tibial rigidity and shape properties in archaeological and fossil samples. Am J Phys Anthropol 2009. © 2009 Wiley‐Liss, Inc. 相似文献
915.
Ji-Seon Kim Sung Joon Kim Jong-Wan Park Kyong Soo Park Yang-Sook Chun 《Biochemical and biophysical research communications》2009,379(4):1048-1053
Aryl hydrocarbon receptor nuclear translocator (ARNT) has been known to participate in cellular responses to xenobiotic and hypoxic stresses, as a common partner of aryl hydrocarbon receptor and hypoxia inducible factor-1/2α. Recently, it was reported that ARNT is essential for adequate insulin secretion in response to glucose input and that its expression is downregulated in the pancreatic islets of diabetic patients. In the present study, the authors addressed the mechanism by which ARNT regulates insulin secretion in the INS-1 insulinoma cell line. In ARNT knock-down cells, basal insulin release was elevated, but insulin secretion was not further stimulated by a high-glucose challenge. Electrophysiological analyses revealed that glucose-dependent membrane depolarization was impaired in these cells. Furthermore, KATP channel activity and expression were reduced. Of two KATP channel subunits, Kir6.2 was found to be positively regulated by ARNT at the mRNA and protein levels. Based on these results, the authors suggest that ARNT expresses KATP channel and by so doing regulates glucose-dependent insulin secretion. 相似文献
916.
In recent years, quantum dots (Qdot), with their unique physical, chemical, and optical properties, have been used extensively as probes to visualize several cell membrane receptors and extracellular biomolecules. However, Qdot-based intracellular imaging has always been associated with vital lacunas. High affinity between quantum dots may induce serious aggregation in the cytoplasm; as a result, quantum dot aggregates are usually misinterpreted as quantum dot-probed intracellular molecules. Moreover, due to the more viscous nature of the cytoplasm versus the extracellular aqueous media, aggregation issues become more severe during intracellular studies. In this work, we suggest direct nondestructive serotonin imaging in an intact cell using the quantum dot-based immunoassay with a rapid tunable multicolor imaging system based on the acousto-optic tunable filter. Any false-positive intracellular serotonin molecules that appeared due to the aggregation of quantum dots could be completely discriminated from the real intracellular serotonin granules through multicolor cellular imaging. The developed method is quick and has wide applicability in targeting various intracellular proteins, coenzymes, and micronutrients. 相似文献
917.
918.
Mi-Sun Won Namhui Im Soohyun Park Yinglan Jin Kyung-Sook Chung Kiho Lee Hwan Mook Kim Jung Joon Lee Kyeong Lee 《Biochemical and biophysical research communications》2009,385(1):16-571
Hypoxia-inducible factor (HIF)-1 is a therapeutic target in solid tumors. We report the novel benzimidazole analogue AC1-004, obtained from a chemical library using an HRE-dependent cell-based assay in colorectal carcinoma HCT-116 cells. The accumulation of hypoxia-induced HIF-1α was inhibited by compound AC1-004 in various cancer cells, including HCT-116, MDA-MB435, SK-HEP1, and Caki-1. Further, AC1-004 down-regulated VEGF and EPO, target genes of HIF-1, and inhibited in vitro tube formation of HUVEC, suggesting its potential inhibitory activity on angiogenesis. Importantly, AC1-004 was found to regulate the stability of HIF-1α through the Hsp90-Akt pathway, leading to the degradation of HIF-1α. An in vivo antitumor study demonstrated that AC1-004 reduced tumor size significantly (i.e., by 58.6%), without severe side effects. These results suggest the benzimidazole analogue AC1-004 is a novel HIF inhibitor that targets HIF-1α via the Hsp90-Akt pathway, and that it can be used as a new lead in developing anticancer drugs. 相似文献
919.
Sung Il Kim Joon Hyeok Kwak Hee-Jun Na Jin Kuk Kim Yan Ding Mary E. Choi 《The Journal of biological chemistry》2009,284(33):22285-22296
Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that signals through the interaction of type I (TβRI) and type II (TβRII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-β1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-β1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with TβRI. TGF-β1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of TβRI is crucial for its interaction with TAK1. Both TβRI-mediated TAK1 phosphorylation and TGF-β1-induced TAK1 phosphorylation do not require kinase activity of TβRI. Moreover, TβRI-mediated TAK1 phosphorylation correlates with the degree of its association with TβRI and requires kinase activity of TAK1. TAB1 does not interact with TGF-β receptors, but TAB1 is indispensable for TGF-β1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-β1-induced interaction of TβRI and TβRII triggers dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of TβRI.Members of the transforming growth factor-β (TGF-β)3 superfamily are key regulators of various biological processes such as cellular differentiation, proliferation, apoptosis, and wound healing (1, 2). TGF-β1, the prototype of TGF-β family, is a potent inducer of extracellular matrix synthesis and is well established as a central mediator in the final common pathway of fibrosis associated with progressive kidney diseases (3, 4). Upon ligand stimulation, TGF-β type I (TβRI) and type II (TβRII) receptors form heterotetrameric complexes, by which TβRI is phosphorylated in the GS domain and activated. Smad signaling pathway is well established as a canonical pathway induced by TGF-β1 (5, 6). Receptor-regulated Smads (Smad2 and Smad3) are recruited and activated by the activated TβRI. The phosphorylation in the GS domain (7) and L45 loop (8) of TβRI are crucial for its interaction with receptor-regulated Smads. After phosphorylation, receptor-regulated Smads are rapidly dissociated from TβRI and interact with common Smad (Smad4) followed by nuclear translocation. In addition to the Smad pathway, a recently emerging body of evidence has demonstrated that TGF-β1 also induces various Smad-independent signaling pathways (9–17) by which mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK) (18, 19), p38 MAPK (20–22), and extracellular signal-regulated kinase 1/2 (23, 24) can be activated by TGF-β1.TAK1, initially identified as a MAPK kinase kinase 7 (MKKK7 or MAP3K7) in the TGF-β signaling pathway (11, 12), also can be activated by environmental stress (25), proinflammatory cytokines such as IL-1 and TNF-α (26, 27) and lipopolysaccharide (28). For TAK1 activation, phosphorylation at Thr-187 and Ser-192 in the activation loop of TAK1 is essentially required (29–31). TAK1 can transduce signals to several downstream signaling cascades, including the MAPK kinase (MKK) 4/7-JNK cascade, MKK3/6-p38 MAPK cascade, and nuclear factor κB (NF-κB)-inducing kinase-IκB kinase cascade (26–28). A recent report has shown that TAK1 is also activated by agonists of AMP-activated kinase (AMPK) and ischemia, which in turn activates the LKB1/AMPK pathway, a pivotal energy-sensor pathway (32). TAK1 is also involved in Wnt signaling (33). We and others have previously demonstrated that TAK1 is a major mediator of TGF-β1-induced type I collagen and fibronectin expression through activation of the MKK3-p38 MAPK and MKK4-JNK signaling cascades, respectively (34–37). Furthermore, increased expression and activation of TAK1 enhance p38 phosphorylation and promote interstitial fibrosis in the myocardium from 9-day-old TAK1 transgenic mice (37). These data implicate a crucial role of TAK1 in extracellular matrix production and tissue fibrosis. TAK1 is also implicated in regulation of cell cycle (38), cell apoptosis (39–41), and the Smad signaling pathway (42–44). Thus, TAK1 may function as an important regulator and mediator of TGF-β1-induced Smad-dependent and Smad-independent signaling pathways.It has been demonstrated that TAK1 can be activated by the interaction with TAK1-binding protein 1 (TAB1) by in vitro binding assays and in overexpression studies (29–31); however, it is not clear whether TAB1 plays a crucial role in ligand-induced TAK1 activation. In embryonic fibroblasts from TAB1 null mice, IL-1 and TNF-α could induce TAK1-mediated NF-κB and JNK activation (45). TAK1 activation induced by TNF-α, IL-1, and T-cell receptor requires TAB2 or its homologous protein TAB3 (46–50). Although many questions still remain, much progress has been made in understanding the activation mechanism of TAK1 by inflammatory cytokines (46, 47, 51–53). Ligand binding of IL-1 receptor (IL-1R) results in recruitment of MyD88, which serves as an adaptor for IL-1 receptor-associated kinase (IRAK) 1 and 4. Subsequently IRAK1 is hyperphosphorylated and induces interaction with TNF-α receptor-associated factor 6 (TRAF6), resulting in TRAF6 oligomerization. After oligomerization of TRAF6, IRAK1-TRAF6 complex is dissociated from the receptor and associated with TAK1, which is mediated by TAB2 (or TAB3). In this process polyubiquitination of TRAF6 by Ubc13/Uev1A is thought to be critical for the association with TAB2 (or TAB3), which links TAK1 activation (46, 54, 55). In the case of TNF-α stimulation, TNF-α receptors form trimers and recruit adaptor proteins, TRAF2/5, and receptor-interacting protein 1 on the membrane. Ubc13/Uev1A- and TRAF2-dependent polyubiquitination of receptor-interacting protein 1 induce association of TAB2 (or TAB3), which then activates TAK1. Thus, TAB2 is required for ubiquitin-dependent activation of TAK1 by TRAFs. On the other hand, it has been demonstrated that hematopoietic progenitor kinase 1 plays a role as an upstream mediator of TGF-β-induced TAK1 activation, which in turn activates the MKK4-JNK signaling cascade in 293T cells (56, 57). Besides hematopoietic progenitor kinase 1, it has been also suggested that X-linked inhibitor of apoptosis (XIAP) might link TAK1 to TGF-β/BMP receptors through the capability of XIAP to interact with TGF-β/BMP receptors and TAB1 (58). Thus, although various molecules participate in the activation of TAK1, the precise mechanism by which TGF-β1 induces TAK1 activation is incompletely understood. Here, we provide evidence that the association of TAK1 with TGF-β receptors is important for TGF-β1-induced activation of TAK1 in mouse mesangial cells. TGF-β1 stimulation induces interaction of TβRI and TβRII, triggering dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation, independent of receptor kinase activity of TβRI. 相似文献
920.
Khan SA Hamayun M Kim HY Yoon HJ Seo JC Choo YS Lee IJ Kim SD Rhee IK Kim JG 《Biotechnology letters》2009,31(2):283-287
Plant growth-promoting endophytic fungi with gibberellin-producing ability were isolated from the roots of Carex kobomugi Ohwi, a common sand-dune plant, and bioassayed for plant growth-promotion. A new strain, Arthrinium phaeospermum KACC43901, promoted growth of waito-c rice and Atriplex gemelinii. Analysis of its culture filtrate showed the presence of bioactive GA1 (0.5 ng/ml), GA3 (8.8 ng/ml), GA4 (4.7 ng/ml) and GA7 (2.2 ng/ml) along with physiologically inactive GA5 (0.4 ng/ml), GA9 (0.6 ng/ml), GA12 (0.4 ng/ml), GA15 (0.4 ng/ml), GA19 (0.9 ng/ml) and GA24 (1.8 ng/ml). The fungal isolate was identified through sequence homology and phylogenetic analysis of 18S rDNA (internal
transcribed region).
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献