scholarly journals Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28

2000 ◽  
Vol 348 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Linda HOWARD ◽  
Rose A. MACIEWICZ ◽  
Carl P. BLOBEL
Keyword(s):  
Cell Surface ◽  
Secretory Pathway ◽  
Active Form ◽  
Catalytic Site ◽  
Mouse Lung ◽  
Mutant Form ◽  
Sequence Comparisons ◽  
Mouse Tissues ◽  
Catalytically Active ◽  
Alternatively Spliced

The metalloprotease disintegrins are a family of membrane-anchored glycoproteins with diverse functions in fertilization, myoblast fusion, neurogenesis and protein ectodomain shedding. Here we report a cDNA sequence, encoding a metalloprotease disintegrin, termed ADAM28 (‘a disintegrin and metalloprotease 28’), which was cloned from mouse lung. From protein sequence comparisons, ADAM28 is more closely related to snake venom metalloproteases (SVMPs) than to other ADAMs, and hence may cleave similar substrates to SVMPs, perhaps including components of the extracellular matrix. Northern blot analysis of selected mouse tissues revealed that ADAM28 is expressed highly and in alternatively spliced forms in the epididymis, suggesting a possible role in sperm maturation, and at lower levels in lung. The intracellular maturation of ADAM28 expressed in COS-7 cells resembles that of other ADAMs, in that ADAM28 is made as a precursor and processed to a mature form in a late Golgi compartment of the secretory pathway. Most or all of the mature, and thus presumably catalytically active, form of ADAM28 in COS-7 cells is accessible to cell surface trypsinization, suggesting that ADAM28 functions mainly on the cell surface. A mutation converting the catalytic-site glutamate residue into alanine abolishes pro-domain removal, even though this mutant form of ADAM28 can be transported to the cell surface in a manner similar to the wild-type protein. This suggests that pro-domain removal and maturation of ADAM28 may be, at least in part, autocatalytic. This is in contrast with several other ADAMs, for which furin-like proprotein convertases are involved in pro-domain removal, and in which a glutamate-to-alanine mutation in the catalytic site does not alter pro-domain removal.

scholarly journals Activation and selectivity of OTUB-1 and OTUB-2 deubiquitinylases

2020 ◽  
Vol 295 (20) ◽  
pp. 6972-6982
Author(s):  
Dakshinamurthy Sivakumar ◽  
Vikash Kumar ◽  
Michael Naumann ◽  
Matthias Stein
Keyword(s):  
Active Site ◽  
Electrostatic Interactions ◽  
Active Form ◽  
Cysteine Proteases ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Catalytic Site ◽  
Dynamics Simulation ◽  
Active Site Residues ◽  
Catalytically Active

The ovarian tumor domain (OTU) deubiquitinylating cysteine proteases OTUB1 and OTUB2 (OTU ubiquitin aldehyde binding 1 and 2) are representative members of the OTU subfamily of deubiquitinylases. Deubiquitinylation critically regulates a multitude of important cellular processes, such as apoptosis, cell signaling, and growth. Moreover, elevated OTUB expression has been observed in various cancers, including glioma, endometrial cancer, ovarian cancer, and breast cancer. Here, using molecular dynamics simulation approaches, we found that both OTUB1 and OTUB2 display a catalytic triad characteristic of proteases but differ in their configuration and protonation states. The OTUB1 protein had a prearranged catalytic site, with strong electrostatic interactions between the active-site residues His265 and Asp267. In OTUB2, however, the arrangement of the catalytic triad was different. In the absence of ubiquitin, the neutral states of the catalytic-site residues in OTUB2 were more stable, resulting in larger distances between these residues. Only upon ubiquitin binding did the catalytic triad in OTUB2 rearrange and bring the active site into a catalytically feasible state. An analysis of water access channels revealed only a few diffusion trajectories for the catalytically active form of OTUB1, whereas in OTUB2 the catalytic site was solvent-accessible, and a larger number of water molecules reached and left the binding pocket. Interestingly, in OTUB2, the catalytic residues His224 and Asn226 formed a stable hydrogen bond. We propose that the observed differences in activation kinetics, protonation states, water channels, and active-site accessibility between OTUB1 and OTUB2 may be relevant for the selective design of OTU inhibitors.

scholarly journals A recombinant human ‘mini’-hexokinase is catalytically active and regulated by hexose 6-phosphates

1992 ◽  
Vol 285 (1) ◽  
pp. 193-199 ◽  
Author(s):  
M Magnani ◽  
M Bianchi ◽  
A Casabianca ◽  
V Stocchi ◽  
A Daniele ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Active Form ◽  
Evolutionary Relationship ◽  
Product Inhibition ◽  
Catalytic Site ◽  
Type I ◽  
Regulatory Site ◽  
Allosteric Site ◽  
Reading Frame ◽  
Catalytically Active

Mammalian hexokinase type I is a 100 kDa enzyme that has been considered to be evolved from an ancestral 50 kDa yeast-type hexokinase, insensitive to product inhibition, by gene duplication and fusion. According to this model, and based on many experimental data, the catalytic site is associated with the C-terminal half of the enzyme, although an allosteric site for the binding of glucose 6-phosphate could be present on the N-terminal half of the molecule. We have isolated a cDNA clone of hexokinase from a lambda gt11 human placenta library comprising 2658 bp, containing a single open reading frame of 1893 nucleotides, which encodes a truncate form of hexokinase starting from asparagine-287 to the terminal serine-917. This clone was further digested with restriction enzyme NcoI to obtain almost only the C-terminal half of human hexokinase starting from methionine-455 to the terminal amino acid and was overexpressed in active form in Escherichia coli and purified by ion-exchange h.p.l.c. The overexpressed ‘mini’-hexokinase was found not only to catalyse glucose phosphorylation, but also to be inhibited by glucose 6-phosphate and other mono- and bis-phosphate sugars exactly like the complete mammalian enzyme. These results suggest that the C-terminal half of human hexokinase, in addition to the catalytic site, also contains the regulatory site and that the evolutionary relationship between the hexokinases should be reconsidered by including the appearance of a regulatory site before the gene duplication.

scholarly journals Deletion of the Gene Encoding Proprotein Convertase 5/6 Causes Early Embryonic Lethality in the Mouse

2006 ◽  
Vol 26 (1) ◽  
pp. 354-361 ◽  
Author(s):  
Rachid Essalmani ◽  
Josée Hamelin ◽  
Jadwiga Marcinkiewicz ◽  
Ann Chamberland ◽  
Majambu Mbikay ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Essential Gene ◽  
Proprotein Convertase ◽  
Gene Encoding ◽  
Reverse Transcription Pcr ◽  
Early Embryonic Lethality ◽  
Mouse Tissues ◽  
Viral Glycoproteins ◽  
Alternatively Spliced

ABSTRACT PC5 belongs to the proprotein convertase family and activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. These precursors include prohormones, proreceptors, growth factors, adhesion molecules, and viral glycoproteins. The Pcsk5 gene encodes two alternatively spliced isoforms, the soluble PC5A and transmembrane PC5B. We have carefully analyzed the expression of PC5 in the mouse during development and in adulthood by in situ hybridization, as well as in mouse tissues and various cell lines by quantitative reverse transcription-PCR. The data show that adrenal cortex and intestine are the richest sources of PC5A and PC5B, respectively. To better define the specific physiological roles of PC5, we have generated a mouse Pcsk5 Δ4 -deficient allele missing exon 4 that encodes the catalytic Asp173. While Δ4/+ heterozygotes were healthy and fertile, genotyping of progeny obtained from Δ4/+ interbreeding indicated that Δ4/Δ4 embryos died between embryonic days 4.5 and 7.5. These data demonstrate that Pcsk5 is an essential gene.

scholarly journals A Dominant-negative Clathrin Mutant Differentially Affects Trafficking of Molecules with Distinct Sorting Motifs in the Class II Major Histocompatibility Complex (MHC) Pathway

1998 ◽  
Vol 140 (5) ◽  
pp. 1023-1037 ◽  
Author(s):  
Shu-Hui Liu ◽  
Michael S. Marks ◽  
Frances M. Brodsky
Keyword(s):  
Cell Surface ◽  
Secretory Pathway ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Cell Surface Expression ◽  
Dominant Negative Mutant ◽  
Mutant Form ◽  
Sorting Signal ◽  
Sorting Signals

The role of clathrin in intracellular sorting was investigated by expression of a dominant-negative mutant form of clathrin, termed the hub fragment. Hub inhibition of clathrin-mediated membrane transport was established by demonstrating a block of transferrin internalization and an alteration in the intracellular distribution of the cation-independent mannose-6-phosphate receptor. Hubs had no effect on uptake of FITC-dextran, adaptor distribution, organelle integrity in the secretory pathway, or cell surface expression of constitutively secreted molecules. Hub expression blocked lysosomal delivery of chimeric molecules containing either the tyrosine-based sorting signal of H2M or the dileucine-based sorting signal of CD3γ, confirming a role for clathrin-coated vesicles (CCVs) in recognizing these signals and sorting them to the endocytic pathway. Hub expression was then used to probe the role of CCVs in targeting native molecules bearing these sorting signals in the context of HLA–DM and the invariant chain (I chain) complexed to HLA–DR. The distribution of these molecules was differentially affected. Accumulation of hubs before expression of the DM dimer blocked DM export from the TGN, whereas hubs had no effect on direct targeting of the DR–I chain complex from the TGN to the endocytic pathway. However, concurrent expression of hubs, such that hubs were building to inhibitory concentrations during DM or DR–I chain expression, caused cell surface accumulation of both complexes. These observations suggest that both DM and DR–I chain are directly transported to the endocytic pathway from the TGN, DM in CCVs, and DR–I chain independent of CCVs. Subsequently, both complexes can appear at the cell surface from where they are both internalized by CCVs. Differential packaging in CCVs in the TGN, mediated by tyrosine- and dileucine-based sorting signals, could be a mechanism for functional segregation of DM from DR–I chain until their intended rendezvous in late endocytic compartments.

scholarly journals Expression and Functional Assessment of an Alternatively Spliced Extracellular Ca2+-Sensing Receptor in Growth Plate Chondrocytes

Endocrinology ◽  
2005 ◽  
Vol 146 (12) ◽  
pp. 5294-5303 ◽  
Author(s):  
Luis Rodriguez ◽  
Chialing Tu ◽  
Zhiqiang Cheng ◽  
Tsui-Hua Chen ◽  
Daniel Bikle ◽  
...  
Keyword(s):  
Cell Surface ◽  
Growth Plate ◽  
Inositol Phosphate ◽  
Intact Cell ◽  
Chinese Hamster ◽  
Full Length ◽  
Wild Type ◽  
Growth Plate Chondrocytes ◽  
Matrix Gene ◽  
Alternatively Spliced

The extracellular Ca2+-sensing receptor (CaR) plays an essential role in mineral homeostasis. Studies to generate CaR-knockout (CaR−/−) mice indicate that insertion of a neomycin cassette into exon 5 of the mouse CaR gene blocks the expression of full-length CaRs. This strategy, however, allows for the expression of alternatively spliced CaRs missing exon 5 [Exon5(−)CaRs]. These experiments addressed whether growth plate chondrocytes (GPCs) from CaR−/− mice express Exon5(−)CaRs and whether these receptors activate signaling. RT-PCR and immunocytochemistry confirmed the expression of Exon5(−)CaR in growth plates from CaR−/− mice. In Chinese hamster ovary or human embryonic kidney-293 cells, recombinant human Exon5(−)CaRs failed to activate phospholipase C likely due to their inability to reach the cell surface as assessed by intact-cell ELISA and immunocytochemistry. Human Exon5(−)CaRs, however, trafficked normally to the cell surface when overexpressed in wild-type or CaR−/− GPCs. Immunocytochemistry of growth plate sections and cultured GPCs from CaR−/− mice showed easily detectable cell-membrane expression of endogenous CaRs (presumably Exon5(−)CaRs), suggesting that trafficking of this receptor form to the membrane can occur in GPCs. In GPCs from CaR−/− mice, high extracellular [Ca2+] ([Ca2+]e) increased inositol phosphate production with a potency comparable with that of wild-type GPCs. Raising [Ca2+]e also promoted the differentiation of CaR−/− GPCs as indicated by changes in proteoglycan accumulation, mineral deposition, and matrix gene expression. Taken together, our data support the idea that expression of Exon5(−)CaRs may compensate for the loss of full-length CaRs and be responsible for sensing changes in [Ca2+]e in GPCs in CaR−/− mice.

scholarly journals Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

2000 ◽  
Vol 20 (15) ◽  
pp. 5516-5528 ◽  
Author(s):  
Žaklina Strezoska ◽  
Dimitri G. Pestov ◽  
Lester F. Lau
Keyword(s):  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Weight Fraction ◽  
Rnase A ◽  
Mutant Form ◽  
Rrna Processing ◽  
Ribonucleoprotein Complexes ◽  
Nuclear Extracts ◽  
Mouse Tissues ◽  
Mouse Cells

ABSTRACT We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G1 in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1Δ, which lacks 231 amino acids in the N- terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1Δ leads to cell growth arrest in the G1phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1Δ expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1Δ in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.

scholarly journals Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning

2013 ◽  
Vol 304 (1) ◽  
pp. H94-H103 ◽  
Author(s):  
Aude Belliard ◽  
Yoann Sottejeau ◽  
Qiming Duan ◽  
Jessa L. Karabin ◽  
Sandrine V. Pierre
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Ion Transport ◽  
Cell Viability ◽  
Ischemia Reperfusion ◽  
Resistant Mutant ◽  
Surface Expression ◽  
Live Cells ◽  
Mutant Form ◽  
Dependent Manner

Na+,K+-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na+,K+-ATPase α1- and α3-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that α1-cell surface abundance was significantly decreased. Na+,K+-ATPase-activity in crude homogenates and 86Rb+ transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 μM ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKCε-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na+,K+-ATPase activity and surface expression. This model also revealed that Na+,K+-ATPase-mediated 86Rb+ uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na+,K+-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na+,K+-ATPase α1 known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na+,K+-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na+,K+-ATPase may be independent of ion transport.

scholarly journals Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport.

1992 ◽  
Vol 118 (2) ◽  
pp. 285-299 ◽  
Author(s):  
H Liu ◽  
A Bretscher
Keyword(s):  
Cell Surface ◽  
Secretory Pathway ◽  
Synthetic Lethality ◽  
Vesicular Transport ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Apparent Loss ◽  
Abnormal Accumulation ◽  
Late Step ◽  
Actin Cables

Disruption of the yeast tropomyosin gene TPM1 results in the apparent loss of actin cables from the cytoskeleton (Liu, H., and A. Bretscher. 1989. Cell. 57:233-242). Here we show that TPM1 disrupted cells grow slowly, show heterogeneity in cell size, have delocalized deposition of chitin, and mate poorly because of defects in both shmooing and cell fusion. The transit time of alpha-factor induced a-agglutinin secretion to the cell surface is longer than in isogenic wild-type strains, and some of the protein is mislocalized. Many of the TPM1-deleted cells contain abundant vesicles, similar in morphology to late secretory vesicles, but without an abnormal accumulation of intermediates in the delivery of either carboxypeptidase Y to the vacuole or invertase to the cell surface. Combinations of the TPM1 disruption with sec13 or sec18 mutations, which affect early steps in the secretory pathway, block vesicle accumulation, while combinations with sec1, sec4 or sec6 mutations, which affect a late step in the secretory pathway, have no effect on the vesicle accumulation. The phenotype of the TPM1 disrupted cells is very similar to that of a conditional mutation in the MYO2 gene, which encodes a myosin-like protein (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). The myo2-66 conditional mutation shows synthetic lethality with the TPM1 disruption, indicating that the MYO2 and TPM1 gene products may be involved in the same, or parallel function. We conclude that tropomyosin, and by inference actin cables, may facilitate directed vesicular transport of components to the correct location on the cell surface.

scholarly journals Thermal inactivation and chaperonin-mediated renaturation of mitochondrial aspartate aminotransferase

1998 ◽  
Vol 334 (1) ◽  
pp. 219-224 ◽  
Author(s):  
James M. LAWTON ◽  
Shawn DOONAN
Keyword(s):  
Escherichia Coli ◽  
Aspartate Aminotransferase ◽  
Thermal Inactivation ◽  
Active Form ◽  
Active Conformation ◽  
E Coli ◽  
Catalytically Active ◽  
Mitochondrial Aspartate Aminotransferase ◽  
Chaperonin 10 ◽  
Chaperonin 60

Mitochondrial aspartate aminotransferase is inactivated irreversibly on heating. The inactivated protein aggregates, but aggregation is prevented by the presence of the chaperonin 60 from Escherichia coli (GroEL). The chaperonin increases the rate of thermal inactivation in the temperature range 55–65 °C but not at lower temperatures. It has previously been shown [Twomey and Doonan (1997) Biochim. Biophys. Acta 1342, 37–44] that the enzyme switches to a modified, but catalytically active, conformation at approx. 55–60 °C and the present results show that this conformation is recognized by and binds to GroEL. The thermally inactivated protein can be released from GroEL in an active form by the addition of chaperonin 10 from E. coli (GroES)/ATP, showing that inactivation is not the result of irreversible chemical changes. These results suggest that the irreversibility of thermal inactivation is due to the formation of an altered conformation with a high kinetic barrier to refolding rather than to any covalent changes. In the absence of chaperonin the unfolded molecules aggregate but this is a consequence, rather than the cause, of irreversible inactivation.

scholarly journals A murine fer testis-specific transcript (ferT) encodes a truncated Fer protein.

1990 ◽  
Vol 10 (1) ◽  
pp. 146-153 ◽  
Author(s):  
K Fischman ◽  
J C Edman ◽  
G M Shackleford ◽  
J A Turner ◽  
W J Rutter ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Translation System ◽  
Appearance Time ◽  
Acid Protein ◽  
Mouse Tissues ◽  
Free Translation ◽  
Alternatively Spliced ◽  
A Cell ◽  
Testis Cdna ◽  
Carboxy Terminal

A cDNA for a potential tyrosine kinase-encoding mRNA was isolated from a mouse testis cDNA library. In a survey of eight mouse tissues, a transcript of 2.4 kilobases restricted to testis tissue was found. The mRNA encodes a 453-amino-acid protein of 51,383 daltons, the smallest tyrosine kinase protein ever described. RNA synthesized from the cDNA template directs the synthesis of a 51,000-Mr protein in a cell-free translation system. The carboxy-terminal 409 amino acids are 98 and 90% identical to the carboxy halves of the rat and human Fer proteins, respectively. This suggests that the cDNA represents an alternatively spliced testis-specific fer mRNA and is therefore termed by us ferT. On the basis of the appearance time of the fer mRNA in the testis of maturing neonatal mice, we speculate on the role played by this protein in the development of this organ.

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