A novel human small subunit of calpains

2002 ◽  
Vol 362 (2) ◽  
pp. 383-388 ◽  
Author(s):  
Éva SCHÁD ◽  
Attila FARKAS ◽  
Gáspár JÉKELY ◽  
Peter TOMPA ◽  
Peter FRIEDRICH
Keyword(s):  
Expressed Sequence Tag ◽  
Large Subunit ◽  
Cysteine Proteases ◽  
Small Subunit ◽  
Regulatory Subunit ◽  
Intronless Gene ◽  
Functional Equivalent

Typical calpains are heterodimeric cysteine proteases which have distinct large catalytic subunits (80kDa) but share a common small regulatory subunit (30kDa; css1). Here we report the identification, cloning and characterization of a novel human small subunit (css2) encoded by an intronless gene, capns2, located on chromosome 16. This new protein displays 73% sequence identity within the Ca2+-binding region but lacks two oligo-Gly stretches characteristic of the N-terminal domain of the conventional small subunit. css2 appears to be the functional equivalent of the conventional small subunit in vitro in that it helps the large subunit fold into the active conformation of similar Ca2+ sensitivity when the two proteins are co-expressed in Escherichia coli. The purification of various chimaeric rat 80kDa—human css2 constructs, on the other hand, shows that css2 binds the large subunit much more weakly than css1. Further, it does not undergo the autolytic conversion typical of the classical small subunit. The expression of this protein in vivo, as assessed from its appearance in expressed sequence tag clones, is rather limited, making it an example of a tissue-specific, rather than ubiquitous, small subunit.

scholarly journals Investigation of in Vivo Roles of the C-terminal Tails of the Small Subunit (ββ′) of Saccharomyces cerevisiae Ribonucleotide Reductase

2013 ◽  
Vol 288 (20) ◽  
pp. 13951-13959 ◽  
Author(s):  
Yan Zhang ◽  
Xiuxiang An ◽  
JoAnne Stubbe ◽  
Mingxia Huang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Small Subunit ◽  
Cluster Assembly ◽  
E Coli ◽  
Viability Assay ◽  
Docking Model

The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2III-Y•) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr356 in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ′), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr376 nor β′-Tyr323 (Tyr356 equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for RT, whereas Tyr323 in β′ is not. Although the C-terminal tail of β′ is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β′ C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β′ interface.

scholarly journals A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action

2011 ◽  
Vol 22 (1) ◽  
pp. 141-152 ◽  
Author(s):  
Xiao-Wei Chen ◽  
Dara Leto ◽  
Tingting Xiong ◽  
Genggeng Yu ◽  
Alan Cheng ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Small Gtpase ◽  
Regulatory Subunit ◽  
Hydrolysis Of ◽  
Identification And Characterization

Insulin stimulates glucose transport in muscle  and adipose tissue by translocation of glucose transporter 4 (GLUT4) to the plasma membrane. We previously reported that activation of the small GTPase RalA downstream of PI 3-kinase plays a critical role in this process by mobilizing the exocyst complex for GLUT4 vesicle targeting in adipocytes. Here we report the identification and characterization of a Ral GAP complex (RGC) that mediates the activation of RalA downstream of the PI 3-kinase/Akt pathway. The complex is composed of an RGC1 regulatory subunit and an RGC2 catalytic subunit (previously identified as AS250) that directly stimulates the guanosine triphosphate hydrolysis of RalA. Knockdown of RGC proteins leads to increased RalA activity and glucose uptake in adipocytes. Insulin inhibits the GAP complex through Akt2-catalyzed phosphorylation of RGC2 in vitro and in vivo, while activated Akt relieves the inhibitory effect of RGC proteins on RalA activity. The RGC complex thus connects PI 3-kinase/Akt activity to the transport machineries responsible for GLUT4 translocation.

scholarly journals Interaction between Subunits of Heterodimeric Splicing Factor U2AF Is Essential In Vivo

1998 ◽  
Vol 18 (4) ◽  
pp. 1765-1773 ◽  
Author(s):  
David Z. Rudner ◽  
Roland Kanaar ◽  
Kevin S. Breger ◽  
Donald C. Rio
Keyword(s):  
Amino Acid ◽  
Large Subunit ◽  
Critical Role ◽  
Small Subunit ◽  
Splicing Factor ◽  
Interaction Domain ◽  
Recessive Lethal ◽  
Acid Fragment

ABSTRACT The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary factor), plays a critical role in 3′ splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia colicopurification assay to map the domain on the DrosophilaU2AF large subunit (dU2AF50) that interacts with theDrosophila small subunit (dU2AF38). A 28-amino-acid fragment on dU2AF50 that is both necessary and sufficient for interaction with dU2AF38 was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF50 point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF50 allele. A mutation that completely abolished interaction with dU2AF38 was incapable of complementation, whereas dU2AF50 mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF50 allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF50 recessive lethal allele. These data indicate thatDrosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro.

scholarly journals Calpain-mediated protein targets in cardiac mitochondria following ischemia–reperfusion

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Ling Li ◽  
Jeremy Thompson ◽  
Ying Hu ◽  
Edward J. Lesnefsky ◽  
Belinda Willard ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Mitochondrial Permeability Transition ◽  
Ischemia Reperfusion ◽  
Large Subunit ◽  
Cardiac Injury ◽  
Cysteine Proteases ◽  
Complex Iii ◽  
Regulatory Subunit ◽  
Protein Targets

AbstractCalpain 1 and 2 (CPN1/2) are calcium-dependent cysteine proteases that exist in cytosol and mitochondria. Pharmacologic inhibition of CPN1/2 decreases cardiac injury during ischemia (ISC)–reperfusion (REP) by improving mitochondrial function. However, the protein targets of CPN1/2 activation during ISC–REP are unclear. CPN1/2 include a large subunit and a small regulatory subunit 1 (CPNS1). Genetic deletion of CPNS1 eliminates the activities of both CPN1 and CPN2. Conditional cardiomyocyte specific CPNS1 deletion mice were used in the present study to clarify the role of CPN1/2 activation in mitochondrial damage during ISC–REP with an emphasis on identifying the potential protein targets of CPN1/2. Isolated hearts from wild type (WT) or CPNS1 deletion mice underwent 25 min in vitro global ISC and 30 min REP. Deletion of CPNS1 led to decreased cytosolic and mitochondrial calpain 1 activation compared to WT. Cardiac injury was decreased in CPNS1 deletion mice following ISC–REP as shown by the decreased infarct size compared to WT. Compared to WT, mitochondrial function was improved in CPNS1 deletion mice following ischemia–reperfusion as shown by the improved oxidative phosphorylation and decreased susceptibility to mitochondrial permeability transition pore opening. H2O2 generation was also decreased in mitochondria from deletion mice following ISC–REP compared to WT. Deletion of CPNS1 also resulted in less cytochrome c and truncated apoptosis inducing factor (tAIF) release from mitochondria. Proteomic analysis of the isolated mitochondria showed that deletion of CPNS1 increased the content of proteins functioning in regulation of mitochondrial calcium homeostasis (paraplegin and sarcalumenin) and complex III activity. These results suggest that activation of CPN1 increases cardiac injury during ischemia–reperfusion by impairing mitochondrial function and triggering cytochrome c and tAIF release from mitochondria into cytosol.

scholarly journals Rsp5 Ubiquitin-Protein Ligase Mediates DNA Damage-Induced Degradation of the Large Subunit of RNA Polymerase II in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (10) ◽  
pp. 6972-6979 ◽  
Author(s):  
Sylvie L. Beaudenon ◽  
Maria R. Huacani ◽  
Guangli Wang ◽  
Donald P. McDonnell ◽  
Jon M. Huibregtse
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
26S Proteasome ◽  
Regulatory Subunit ◽  
Ubiquitin Protein Ligase

ABSTRACT Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation ofSEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.

scholarly journals Transcriptional and post-transcriptional regulation of ribulose 1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons.

1985 ◽  
Vol 5 (9) ◽  
pp. 2238-2246 ◽  
Author(s):  
J O Berry ◽  
B J Nikolau ◽  
J P Carr ◽  
D F Klessig
Keyword(s):  
Specific Activity ◽  
Large Subunit ◽  
Growth Conditions ◽  
Small Subunit ◽  
Northern Analysis ◽  
Mrna Levels ◽  
Regulation Of Expression ◽  
Bisphosphate Carboxylase

The regulation of expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in 1- through 8-day-old, dark-grown (etiolated) and light-grown amaranth cotyledons. RuBPCase specific activity in light-grown cotyledons increased during this 8-day period to a level 15-fold higher than in dark-grown cotyledons. Under both growth conditions, the accumulation of the LSU and SSU polypeptides was not coordinated. Initial detection of the SSU occurred 1 and 2 days after the appearance of the LSU in light- and dark-grown cotyledons, respectively. Furthermore, although the levels of the LSU were similar in both light- and dark-grown seedlings, the amount of the SSU followed clearly the changes in enzyme activity. Synthesis of these two polypeptides was dramatically different in etiolated versus light-grown cotyledons. In light the synthesis of both subunits was first observed on day 2 and continued throughout the growth of the cotyledons. In darkness the rate of synthesis of both subunits was much lower than in light and occurred only as a burst between days 2 and 5 after planting. However, mRNAs for both subunits were present in etiolated cotyledons at similar levels on days 4 through 7 (by Northern analysis) and were functional in vitro, despite their apparent inactivity in vivo after day 5. In addition, since both LSU and SSU mRNA levels were lower in dark- than in light-grown seedlings, our results indicate that both transcriptional and post-transcriptional controls modulate RuBPCase production in developing amaranth cotyledons.

scholarly journals Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase II-specific role in basal and activated transcription.

1995 ◽  
Vol 15 (3) ◽  
pp. 1234-1243 ◽  
Author(s):  
J J Kang ◽  
D T Auble ◽  
J A Ranish ◽  
S Hahn
Keyword(s):  
Transcription Factor ◽  
Large Subunit ◽  
Small Subunit ◽  
Specific Factor ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Pol Ii ◽  
Conserved Regions

To probe the structure and function of the Saccharomyces cerevisiae general transcription factor TFIIA, we have systematically mutagenized the genes encoding both subunits and analyzed the effects of the mutations both in vivo and in vitro. We found that the central nonconserved region of the large subunit is not essential for function and likely acts as a spacer between the conserved N- and C-terminal regions. Deletion mutagenesis of the large subunit defined a region which is required for TATA binding protein (TBP) interaction. Alanine scanning mutagenesis defined a cluster of four basic residues which are likely required for interaction with DNA in the TBP-DNA complex. Much of the conserved regions of both subunits is required for subunit association, suggesting that these conserved regions fold into compact domains which extensively interact. In vitro transcription performed with extracts from yeast strains with mutations in either the large or the small TFIIA subunit demonstrated that TFIIA stimulates both basal and activated polymerase II (Pol II) transcription. The TFIIA-depleted extracts have normal Pol I and Pol III transcription activity, showing that TFIIA is a Pol II-specific factor. In vivo depletion of TFIIA activity reduced transcription from four different Pol II promoters. Finally, alanine scanning mutagenesis of TFIIA's small subunit has identified at least one mutation which is defective in transcription but which is not defective in subunit association or binding to TBP or TBP-DNA complexes.

Transcriptional and post-transcriptional regulation of ribulose 1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons

1985 ◽  
Vol 5 (9) ◽  
pp. 2238-2246
Author(s):  
J O Berry ◽  
B J Nikolau ◽  
J P Carr ◽  
D F Klessig
Keyword(s):  
Specific Activity ◽  
Large Subunit ◽  
Growth Conditions ◽  
Small Subunit ◽  
Northern Analysis ◽  
Mrna Levels ◽  
Regulation Of Expression ◽  
Bisphosphate Carboxylase

The regulation of expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in 1- through 8-day-old, dark-grown (etiolated) and light-grown amaranth cotyledons. RuBPCase specific activity in light-grown cotyledons increased during this 8-day period to a level 15-fold higher than in dark-grown cotyledons. Under both growth conditions, the accumulation of the LSU and SSU polypeptides was not coordinated. Initial detection of the SSU occurred 1 and 2 days after the appearance of the LSU in light- and dark-grown cotyledons, respectively. Furthermore, although the levels of the LSU were similar in both light- and dark-grown seedlings, the amount of the SSU followed clearly the changes in enzyme activity. Synthesis of these two polypeptides was dramatically different in etiolated versus light-grown cotyledons. In light the synthesis of both subunits was first observed on day 2 and continued throughout the growth of the cotyledons. In darkness the rate of synthesis of both subunits was much lower than in light and occurred only as a burst between days 2 and 5 after planting. However, mRNAs for both subunits were present in etiolated cotyledons at similar levels on days 4 through 7 (by Northern analysis) and were functional in vitro, despite their apparent inactivity in vivo after day 5. In addition, since both LSU and SSU mRNA levels were lower in dark- than in light-grown seedlings, our results indicate that both transcriptional and post-transcriptional controls modulate RuBPCase production in developing amaranth cotyledons.

scholarly journals Analysis of Mutant Phenotypes and Splicing Defects Demonstrates Functional Collaboration between the Large and Small Subunits of the Essential Splicing Factor U2AF In Vivo

2005 ◽  
Vol 16 (2) ◽  
pp. 584-596 ◽  
Author(s):  
Christopher J. Webb ◽  
Sujata Lakhe-Reddy ◽  
Charles M. Romfo ◽  
Jo Ann Wise
Keyword(s):  
Splice Site ◽  
Large Subunit ◽  
Small Subunit ◽  
Splicing Factor ◽  
In Vitro Assays ◽  
Growth Defect ◽  
Mutant Phenotypes ◽  
Site Recognition

The heterodimeric splicing factor U2AF plays an important role in 3′ splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3′ splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AFLGare essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3′ splice site recognition, we sought introns for which the spU2AFLGand spU2AFSMmake distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3′ pyrimidine tract. These and other studies performed in fission yeast support a model for 3′ splice site recognition in which the two subunits of U2AF functionally collaborate in vivo.

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