The Molecular Chaperone, ClpA, Has a Single High Affinity Peptide Binding Site per Hexamer

The physiological functions of hyaluronan (HA) in the extracellular matrix of vertebrate tissues involve a range of specific protein interactions. In this study, the interaction of HA with the Link module from TSG-6 (Link_TSG6) and G1 domain of aggrecan (G1), were investigated by a biophysical analysis of translational diffusion in dilute solution using confocal fluorescence recovery after photobleaching (confocal FRAP). Both Link_TSG6 and G1 were shown to bind to polymeric HA and these interactions could be competed with HA(8) and HA(10) oligosaccharides, respectively. Equilibrium experiments showed that the binding affinity of Link_TSG6 to HA was maximal at pH 6.0, and reduced dramatically above and below this pH. In contrast, G1 had maximum binding at pH 7.0-8.0 and moderate to strong binding affinity over a much broader pH range (5.5-8.0). The K(D) determined for Link_TSG6 binding to HA showed a 100-fold increase in binding affinity between pH 7.4 and 6.0, whereas G1 showed a 75-fold decrease in binding affinity over the same pH range. The sharp difference observed in their pH binding suggests that pH controls the physiological function of TSG-6, with a low affinity for HA at neutral pH, but with increased affinity as the pH falls below pH 7. TSG-6 and aggrecan interact with HA through structurally homologous domains and the difference in pH-dependent binding can be understood in terms of differences in the presence and topographical distribution of key regulatory amino acids in Link_TSG6 and in the related tandem Link domains in aggrecan G1.

Streptokinase (SK) activates human fibrinolysis by inducing non-proteolytic activation of the serine proteinase zymogen, plasminogen (Pg), in the SK.Pg* catalytic complex. SK.Pg* proteolytically activates Pg to plasmin (Pm). SK-induced Pg activation is enhanced by lysine-binding site (LBS) interactions with kringles on Pg and Pm, as evidenced by inhibition of the reactions by the lysine analogue, 6-aminohexanoic acid. Equilibrium binding analysis and [Lys]Pg activation kinetics with wild-type SK, carboxypeptidase B-treated SK, and a COOH-terminal Lys414 deletion mutant (SKDeltaK414) demonstrated a critical role for Lys414 in the enhancement of [Lys]Pg and [Lys]Pm binding and conformational [Lys]Pg activation. The LBS-independent affinity of SK for [Glu]Pg was unaffected by deletion of Lys414. By contrast, removal of SK Lys414 caused 19- and 14-fold decreases in SK affinity for [Lys]Pg and [Lys]Pm binding in the catalytic mode, respectively. In kinetic studies of the coupled conformational and proteolytic activation of [Lys]Pg, SKDeltaK414 exhibited a corresponding 17-fold affinity decrease for formation of the SKDeltaK414.[Lys]Pg* complex. SKDeltaK414 binding to [Lys]Pg and [Lys]Pm and conformational [Lys]Pg activation were LBS-independent, whereas [Lys]Pg substrate binding and proteolytic [Lys]Pm generation remained LBS-dependent. We conclude that binding of SK Lys414 to [Lys]Pg and [Lys]Pm kringles enhances SK.[Lys]Pg* and SK.[Lys]Pm catalytic complex formation. This interaction is distinct structurally and functionally from LBS-dependent Pg substrate recognition by these complexes.

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

Electrocardiographic features of patients with COVID-19: One year of unexpected manifestations

Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface.

CaV1.2 interacts with the Ca(2+) sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α1C. Ca(2+)/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca(2+) ions (Ca(2+)-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Cav1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaM altered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that the NT is not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.

The linkage of heterodimeric (alpha/beta) integrin receptors with their extracellular matrix ligands and intracellular actin cytoskeleton is a fundamental step for controlling cell adhesion and migration. Binding of the actin-linking protein, talin, to integrin beta cytoplasmic tails (CTs) induces high affinity ligand binding (integrin activation), whereas binding of another actin-linking protein, filamin, to the integrin beta CTs negatively regulates this process by blocking the talin-integrin interaction. Here we show structurally that migfilin, a novel cytoskeletal adaptor highly enriched in the integrin adhesion sites, strongly interacts with the same region in filamin where integrin beta CTs bind. We further demonstrate that the migfilin interaction dissociates filamin from integrin and promotes the talin/integrin binding and integrin activation. Migfilin thus acts as a molecular switch to disconnect filamin from integrin for regulating integrin activation and dynamics of extracellular matrix-actin linkage.

The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to the alpha(3)beta(3) hexagon of F(1). To quantitatively characterize binding of b subunit to the F(1) alpha(3)beta(3) hexagon, we developed fluorimetric assays in which wild-type F(1), or F(1) enzymes containing introduced Trp residues, were titrated with a soluble portion of the b subunit (b(ST34-156)). With five different F(1) enzymes, K(d)(b(ST34-156)) ranged from 91 to 157 nm. Binding was strongly Mg(2+)-dependent; in EDTA buffer, K(d)(b(ST34-156)) was increased to 1.25 microm. The addition of the cytoplasmic portion of the b subunit increases the affinity of binding of delta subunit to delta-depleted F(1). The apparent K(d)(b(ST34-156)) for this effect was increased from 150 nm in Mg(2+) buffer to 1.36 microm in EDTA buffer. This work demonstrates quantitatively how binding of the cytoplasmic portion of the b subunit directly to F(1) contributes to stator resistance and emphasizes the importance of Mg(2+) in stator interactions.

Pulmonary Hypertension: From an Orphan Disease to a Public Health Problem

The recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance. Non-catalytic carbohydrate-binding modules (CBMs) play a key role in this degradative process by targeting hydrolytic enzymes to their cognate substrate within the complex milieu of polysaccharides that comprise the plant cell wall. Family 29 CBMs have, thus far, only been found in an extracellular multienzyme plant cell wall-degrading complex from the anaerobic fungus Piromyces equi, where they exist as a CBM29-1:CBM29-2 tandem. Here we present both the structure of the CBM29-1 partner, at 1.5 A resolution, and examine the importance of hydrophobic stacking interactions as well as direct and solvent-mediated hydrogen bonds in the binding of CBM29-2 to different polysaccharides. CBM29 domains display unusual binding properties, exhibiting specificity for both beta-manno- and beta-gluco-configured ligands such as mannan, cellulose, and glucomannan. Mutagenesis reveals that "stacking" of tryptophan residues in the n and n+2 subsites plays a critical role in ligand binding, whereas the loss of tyrosine-mediated stacking in the n+4 subsite reduces, but does not abrogate, polysaccharide recognition. Direct hydrogen bonds to ligand, such as those provided by Arg-112 and Glu-78, play a pivotal role in the interaction with both mannan and cellulose, whereas removal of water-mediated interactions has comparatively little effect on carbohydrate binding. The interactions of CBM29-2 with the O2 of glucose or mannose contribute little to binding affinity, explaining why this CBM displays dual gluco/manno specificity.

Impaired Autophagy Triggers Chronic Pancreatitis: Lessons From Pancreas-Specific Atg5 Knockout Mice

The multipotentiality and self-renewal ability of stem cells are controlled by intrinsic genetic pathways that are subject to regulation by extrinsic signals emanating from the stem cell niche. The stem cell niche provides a microenvironment composed of cellular structures or extracellular matrix in which stem cells are maintained as undifferentiated (1Li L. Xie T. Annu. Rev. Cell Dev. Biol. 2005; 21: 605-631Crossref PubMed Scopus (942) Google Scholar, 2Lin H. Nat. Rev. Genet. 2002; 3: 931-940Crossref PubMed Scopus (305) Google Scholar, 3Scadden D.T. Nature. 2006; 441: 1075-1079Crossref PubMed Scopus (1508) Google Scholar, 4Spradling A. Drummond-Barbosa D. Kai T. Nature. 2001; 414: 98-104Crossref PubMed Scopus (1215) Google Scholar, 5Watt F.M. Hogan B.L. Science. 2000; 287: 1427-1430Crossref PubMed Scopus (1474) Google Scholar, 6Fuchs E. Tumbar T. Guasch G. Cell. 2004; 116: 769-778Abstract Full Text Full Text PDF PubMed Scopus (1481) Google Scholar). The concept of "the stem cell niche" was first proposed in studies of the HSC 2The abbreviations used are:HSChematopoietic stem cellGSCgerm line stem cellBMbone marrowCARCXCL12-abundant reticularCBscystoblastsESCescort stem cellCPscyst progenitorsAng-1angiopoietin-1.2The abbreviations used are:HSChematopoietic stem cellGSCgerm line stem cellBMbone marrowCARCXCL12-abundant reticularCBscystoblastsESCescort stem cellCPscyst progenitorsAng-1angiopoietin-1. (7Schofield R. Blood Cells. 1978; 4: 7-25PubMed Google Scholar); however, in vivo evidence of its existence was first shown in the Drosophila GSC (8Cox D.N. Chao A. Baker J. Chang L. Qiao D. Lin H. Genes Dev. 1998; 12: 3715-3727Crossref PubMed Scopus (802) Google Scholar, 9King F.J. Lin H. Development (Camb.). 1999; 126: 1833-1844PubMed Google Scholar, 10Xie T. Spradling A.C. Science. 2000; 290: 328-330Crossref PubMed Scopus (620) Google Scholar). 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In this review, our focus is on comparing Drosophila GSC niches and mouse HSC niches (two of the best characterized niches). By such comparison, we hope to provide some common principles of stem cell niches that will be useful in other tissue stem cell niche studies. hematopoietic stem cell germ line stem cell bone marrow CXCL12-abundant reticular cystoblasts escort stem cell cyst progenitors angiopoietin-1. hematopoietic stem cell germ line stem cell bone marrow CXCL12-abundant reticular cystoblasts escort stem cell cyst progenitors angiopoietin-1. In recent years, remarkable progress has been made in the identification and characterization of the stem cell niches in invertebrate systems (10Xie T. Spradling A.C. Science. 2000; 290: 328-330Crossref PubMed Scopus (620) Google Scholar, 17Tulina N. Matunis E. Science. 2001; 294: 2546-2549Crossref PubMed Scopus (506) Google Scholar, 18Kiger A.A. Jones D.L. Schulz C. Rogers M.B. Fuller M.T. 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These advantages facilitated identification of the cellular components of the stem cell niche and definition of the molecular basis of physical interaction between stem cells and their niches (23Song X. Xie T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14813-14818Crossref PubMed Scopus (184) Google Scholar, 24Yamashita Y.M. Jones D.L. Fuller M.T. Science. 2003; 301: 1547-1550Crossref PubMed Scopus (595) Google Scholar) and revealed key niche signals involved in stem cell regulation (18Kiger A.A. Jones D.L. Schulz C. Rogers M.B. Fuller M.T. Science. 2001; 294: 2542-2545Crossref PubMed Scopus (538) Google Scholar, 25LaFever L. Drummond-Barbosa D. Science. 2005; 309: 1071-1073Crossref PubMed Scopus (248) Google Scholar, 26Szakmary A. Cox D.N. Wang Z. Lin H. Curr. Biol. 2005; 15: 171-178Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 27Xie T. Spradling A.C. Cell. 1998; 94: 251-260Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, 28Yamashita Y.M. 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Two HSC niches have been proposed in murine BM, an osteoblastic niche and a vascular niche, in which osteoblasts and vascular endothelial cells have been demonstrated as major components, respectively (34Zhang J. Niu C. Ye L. Huang H. He X. Tong W.G. Ross J. Haug J. Johnson T. Feng J.Q. Harris S. Wiedemann L.M. Mishina Y. Li L. Nature. 2003; 425: 836-841Crossref PubMed Scopus (2385) Google Scholar, 35Calvi L.M. Adams G.B. Weibrecht K.W. Weber J.M. Olson D.P. Knight M.C. Martin R.P. Schipani E. Divieti P. Bringhurst F.R. Milner L.A. Kronenberg H.M. Scadden D.T. Nature. 2003; 425: 841-846Crossref PubMed Scopus (2799) Google Scholar, 36Arai F. Hirao A. Ohmura M. Sato H. Matsuoka S. Takubo K. Ito K. Koh G.Y. Suda T. Cell. 2004; 118: 149-161Abstract Full Text Full Text PDF PubMed Scopus (1540) Google Scholar, 37Nilsson S.K. Johnston H.M. Whitty G.A. Williams B. Webb R.J. Denhardt D.T. Bertoncello I. Bendall L.J. Simmons P.J. Haylock D.N. 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Immunity. 2006; 25: 977-988Abstract Full Text Full Text PDF PubMed Scopus (1693) Google Scholar). Drosophila ovary and testis provide attractive models for stem cell niche studies. In Drosophila ovary, cap cells, a specific type of somatic cell located at the tip of the germarium in the ovary, function as the niche for GSCs. An E-cadherin/β-catenin-formed cell-cell adhesion junction mediates the physical interaction between GSCs and their niche cells (23Song X. Xie T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14813-14818Crossref PubMed Scopus (184) Google Scholar, 46Song X. Zhu C.H. Doan C. Xie T. Science. 2002; 296: 1855-1857Crossref PubMed Scopus (389) Google Scholar), ensuring stem cell control by niche signals. Normally, asymmetric division of stem cells results in two daughter cells with different fates: one daughter cell attaches to the niche and is maintained as a stem cell (self-renewal), whereas the other daughter cell leaves the niche and develops into a cystoblast (1Li L. Xie T. Annu. Rev. Cell Dev. Biol. 2005; 21: 605-631Crossref PubMed Scopus (942) Google Scholar, 4Spradling A. Drummond-Barbosa D. Kai T. Nature. 2001; 414: 98-104Crossref PubMed Scopus (1215) Google Scholar). CBs undergo four incomplete cell divisions to form an interconnected 2–16-cell germ line cyst. In addition to the GSC, another type of cell called the ESC also attaches to cap cells interspersed between the GSCs. Together with cap cells, ESCs encapsulate GSCs to separate GSCs from their differentiated daughter cells. When the GSCs divide and detach from the niche to produce CBs and cyst cells, the ESCs also proliferate and differentiate to produce more escort cells. The escort cells expand and continue to encapsulate CBs and cysts during the process of cyst formation and are finally replaced by follicle cells after the 16-cell germ line cyst stage (Fig. 1a). Interestingly, the newly formed cysts (prior to the eight-cell cyst stage, while still encapsulated by the escort cell) can revert to the stem cell state under certain circumstances, suggesting that they may still retain (albeit limited) stem cell properties (47Kai T. Spradling A. Nature. 2004; 428: 564-569Crossref PubMed Scopus (267) Google Scholar). A similar niche is also found in Drosophila testis and is composed of hub cells located at the end of the testis (48Le Bras S. Van Doren M. Dev. Biol. 2006; 294: 92-103Crossref PubMed Scopus (96) Google Scholar). Male GSCs surrounding the hub cells are interspersed with CPs (which are counterparts of ESCs in the ovary). Both GSCs and CPs are attached to the hub cells through an adhesion junction. Similar to ESCs in the ovary, proliferation and differentiation of CPs always accompany proliferation and differentiation of GSCs. CPs in the testis produce cyst cells, which encapsulate the gonialblasts (the immediate daughters of male GSCs) during spermatogonium formation (Fig. 1b). The newly formed gonialblasts can also revert to stem cells under certain circumstances, resembling their counterpart cells (CBs and cysts) in the ovary (49Brawley C. Matunis E. Science. 2004; 304: 1331-1334Crossref PubMed Scopus (326) Google Scholar). The role of cap and hub cells in GSC regulation has been well studied, but the contribution of ESCs and their offspring in the ovary and CPs and their progeny in the testis to GSC self-renewal, proliferation, and differentiation is largely unknown. However, it is known that a dialogue between the two types of cells is required for the coordination during organogenesis (30King F.J. Szakmary A. Cox D.N. Lin H. Mol. Cell. 2001; 7: 497-508Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Cap and hub cells provide an attachment point for anchoring GSCs to the niche; they also produce signals that inhibit differentiation but promote self-renewal of stem cells (17Tulina N. Matunis E. Science. 2001; 294: 2546-2549Crossref PubMed Scopus (506) Google Scholar). The stem cell niches in mammals are very complicated because of the complexity of cellular components of mammalian tissues, even though the mechanism for niche regulation is conserved from invertebrate to vertebrate. In mammals, BM tissue is composed of more than eight different hematopoietic cell lineages supported by a network of mesenchymal stromal cells and vascular endothelial cells. The two HSC niches, osteoblastic (34Zhang J. Niu C. Ye L. Huang H. He X. Tong W.G. Ross J. Haug J. Johnson T. Feng J.Q. Harris S. Wiedemann L.M. Mishina Y. Li L. Nature. 2003; 425: 836-841Crossref PubMed Scopus (2385) Google Scholar, 35Calvi L.M. Adams G.B. Weibrecht K.W. Weber J.M. Olson D.P. Knight M.C. Martin R.P. Schipani E. Divieti P. Bringhurst F.R. Milner L.A. Kronenberg H.M. Scadden D.T. Nature. 2003; 425: 841-846Crossref PubMed Scopus (2799) Google Scholar, 36Arai F. Hirao A. Ohmura M. Sato H. Matsuoka S. Takubo K. Ito K. Koh G.Y. Suda T. Cell. 2004; 118: 149-161Abstract Full Text Full Text PDF PubMed Scopus (1540) Google Scholar, 37Nilsson S.K. Johnston H.M. Whitty G.A. Williams B. Webb R.J. Denhardt D.T. Bertoncello I. Bendall L.J. Simmons P.J. Haylock D.N. Blood. 2005; 106: 1232-1239Crossref PubMed Scopus (605) Google Scholar, 38Heissig B. Rafii S. Akiyama H. Ohki Y. Sato Y. Rafael T. Zhu Z. Hicklin D.J. Okumura K. Ogawa H. Werb Z. Hattori K. J. Exp. Med. 2005; 202: 739-750Crossref PubMed Scopus (195) Google Scholar, 39Moore K.A. Curr. Opin. Hematol. 2004; 11: 107-111Crossref PubMed Scopus (56) Google Scholar, 40Taichman R.S. Blood. 2005; 105: 2631-2639Crossref PubMed Scopus (469) Google Scholar, 41Kiel M.J. Yilmaz O.H. Iwashita T. Terhorst C. Morrison S.J. Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2414) Google Scholar, 50Balduino A. Hurtado S.P. Frazao P. Takiya C.M. Alves L.M. Nasciutti L.E. El-Cheikh M.C. Borojevic R. Cell Tissue Res. 2005; 319: 255-266Crossref PubMed Scopus (90) Google Scholar, 51Arai F. Hirao A. Suda T. Trends Cardiovasc. Med. 2005; 15: 75-79Crossref PubMed Scopus (66) Google Scholar, 52Adams G.B. Chabner K.T. Alley I.R. Olson D.P. Szczepiorkowski Z.M. Poznansky M.C. Kos C.H. Pollak M.R. Brown E.M. Scadden D.T. Nature. 2006; 439: 599-603Crossref PubMed Scopus (600) Google Scholar) and vascular (41Kiel M.J. Yilmaz O.H. Iwashita T. Terhorst C. Morrison S.J. Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2414) Google Scholar, 53Kopp H.G. Avecilla S.T. Hooper A.T. Rafii S. Physiology. 2005; 20: 349-356Crossref PubMed Scopus (414) Google Scholar), are found in adult BM tissue. The vascular niche might also exist in fetal hematopoietic tissues such as yolk sac, aorta-gonad-mesonephros region, placenta, liver, and spleen (54Tavian M. Peault B. Exp. Hematol. 2005; 33: 1062-1069Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 55Basto D. Trovisco V. Lopes J.M. Martins A. Pardal F. Soares P. Reis R.M. Acta Neuropathol. 2005; 109: 207-210Crossref PubMed Scopus (84) Google Scholar, 56Gekas C. Dieterlen-Lièvre F. Orkin S.H. Millola H.K.A. Dev. Cell. 2005; 8: 365-375Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar) as well as in adult spleen and liver. Both niches may be critical for HSC self-renewal. Cooperation between these two niches might be required for maintaining normal hematopoietic homeostasis and re-establishing hematopoiesis after injury. Accumulated evidence supports the existence of an osteoblastic niche in BM. 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Quality of Life and Depression in CKD: Improving Hope and Health

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I alpha-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded alpha1-antitrypsin variant (null (Hong Kong)) and of TCRalpha. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded alpha1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent alpha1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of alpha1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has alpha1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.

Melanoma Resistance: A Bright Future for Academicians and a Challenge for Patient Advocates