scholarly journals Identification and cloning of two isoforms of human high-temperature requirement factor A3 (HtrA3), characterization of its genomic structure and comparison of its tissue distribution with HtrA1 and HtrA2

2003 ◽  
Vol 371 (1) ◽  
pp. 39-48 ◽  
Author(s):  
Gui-Ying NIE ◽  
Anne HAMPTON ◽  
Ying LI ◽  
Jock K. FINDLAY ◽  
Lois A. SALAMONSEN
Keyword(s):  
Alternative Splicing ◽  
High Temperature ◽  
Short Form ◽  
Genomic Structure ◽  
Expression Patterns ◽  
Pdz Domain ◽  
Northern Blotting ◽  
Specific Function ◽  
Temperature Requirement ◽  
Long Form

In the present study, we identified an additional member of the human high-temperature requirement factor A (HtrA) protein family, called pregnancy-related serine protease or HtrA3, which was most highly expressed in the heart and placenta. We cloned the full-length sequences of two forms (long and short) of human HtrA3 mRNA, located the gene on chromosome 4p16.1, determined its genomic structure and revealed how the two mRNA variants are produced through alternative splicing. The alternative splicing was also verified by Northern blotting. Four distinct domains were found for the long form HtrA3 protein: (i) an insulin/insulin-like growth factor binding domain, (ii) a Kazal-type S protease-inhibitor domain, (iii) a trypsin protease domain and (iv) a PDZ domain. The short form is identical to the long form except it lacks the PDZ domain. Comparison of all members of human HtrA proteins, including their isoforms, suggests that both isoforms of HtrA3 represent active serine proteases, that they may have different substrate specificities and that HtrA3 may have similar functions to HtrA1. All three HtrA family members showed very different mRNA-expression patterns in 76 human tissues, indicating a specific function for each. Interestingly, both HtrA1 and HtrA3 are highly expressed in the placenta. Identification of the tissue-specific function of each HtrA family member is clearly of importance.

scholarly journals The crystal structure of Mycobacterium tuberculosis high-temperature requirement A protein reveals an autoregulatory mechanism

2018 ◽  
Vol 74 (12) ◽  
pp. 803-809
Author(s):  
Arvind Kumar Gupta ◽  
Debashree Behera ◽  
Balasubramanian Gopal
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
High Temperature ◽  
Catalytic Domain ◽  
Pdz Domain ◽  
Regulatory Pathways ◽  
Inactive Conformation ◽  
Temperature Requirement ◽  
Autoregulatory Mechanism

The crystal structure of Mycobacterium tuberculosis high-temperature requirement A (HtrA) protein was determined at 1.83 Å resolution. This membrane-associated protease is essential for the survival of M. tuberculosis. The crystal structure reveals that interactions between the PDZ domain and the catalytic domain in HtrA lead to an inactive conformation. This finding is consistent with its proposed role as a regulatory protease that is conditionally activated upon appropriate environmental triggers. The structure provides a basis for directed studies to evaluate the role of this essential protein and the regulatory pathways that are influenced by this protease.

The crystal structure of an essential high-temperature requirement protein HtrA1 (Rv1223) from Mycobacterium tuberculosis reveals its unique features

2018 ◽  
Vol 74 (9) ◽  
pp. 906-921 ◽  
Author(s):  
Khundrakpam Herojit Singh ◽  
Savita Yadav ◽  
Deepak Kumar ◽  
Bichitra Kumar Biswal
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
High Temperature ◽  
Protein Quality ◽  
Pdz Domain ◽  
Survival Strategies ◽  
Dimensional Structure ◽  
Protease Domain ◽  
Temperature Requirement

High-temperature requirement A (HtrA) proteins, which are members of the heat-shock-induced serine protease family, are involved in extracytoplasmic protein quality control and bacterial survival strategies under stress conditions, and are associated with the virulence of several pathogens; they are therefore major drug targets. Mycobacterium tuberculosis possesses three putative HtrAs: HtrA1 (Rv1223), HtrA2 (Rv0983) and HtrA3 (Rv0125). Each has a cytoplasmic region, a transmembrane helix and a periplasmic region. Here, the crystal structure of the periplasmic region consisting of a protease domain (PD) and a PDZ domain from an M. tuberculosis HtrA1 mutant (mHtrA1S387A) is reported at 2.7 Å resolution. Although the mHtrA1S387A PD shows structural features similar to those of other HtrAs, its loops, particularly L3 and LA, display different conformations. Loop L3 communicates between the PDs of the trimer and the PDZ domains and undergoes a transition from an active to an inactive conformation, as reported for an equivalent HtrA (DegS). Loop LA, which is responsible for higher oligomer formation owing to its length (50 amino acids) in DegP, is very short in mHtrA1S387A (five amino acids), as in mHtrA2 (also five amino acids), and therefore lacks essential interactions for the formation of higher oligomers. Notably, a well ordered loop known as the insertion clamp in the PDZ domain interacts with the protease domain of the adjacent molecule, which possibly aids in the stabilization of a trimeric functional unit of this enzyme. The three-dimensional structure of mHtrA1S387A presented here will be useful in the design of enzyme-specific antituberculosis inhibitors.

scholarly journals High temperature requirement A1 and fibronectin: two possible players in placental tissue remodelling

2016 ◽  
Author(s):  
G. Tossetta ◽  
C. Avellini ◽  
C. Licini ◽  
S.R. Giannubilo ◽  
M. Castellucci ◽  
...  
Keyword(s):  
Wound Healing ◽  
High Temperature ◽  
Expression Patterns ◽  
Placental Tissue ◽  
Important Process ◽  
Placental Development ◽  
Tissue Remodelling ◽  
Temperature Requirement ◽  
Development And Differentiation ◽  
Growth Zones

High temperature requirement A1 (HtrA1) is a secreted protease involved in placental development. Fibronectin (FN) is involved in important process such as wound healing, cell adhesion and spreading, growth, migration, and differentiation. The purpose of this study was to analyse the expression patterns of HtrA1 in relationship to FN and to the key growth zones of placenta such as mesenchymal villi as well as cell islands and cell columns. We demonstrated that FN and HtrA1 are localized in the placental key growth zones suggesting a pivotal role in maintaining the balance among the molecules involved in the placental development and differentiation.

scholarly journals TheCaenorhabditis elegans unc-32Gene Encodes Alternative Forms of a Vacuolar ATPaseaSubunit

2000 ◽  
Vol 276 (15) ◽  
pp. 11913-11921 ◽  
Author(s):  
Nathalie Pujol ◽  
Claire Bonnerot ◽  
Jonathan J. Ewbank ◽  
Yuji Kohara ◽  
Danielle Thierry-Mieg
Keyword(s):  
Alternative Splicing ◽  
Gene Family ◽  
Synaptic Vesicles ◽  
Expression Patterns ◽  
Cholinergic Neurons ◽  
Specific Function ◽  
Fatal Disease ◽  
Embryonic Lethal ◽  
Genes Encoding ◽  
Acidic Organelles

Eukaryotes possess multiple isoforms of theasubunit of the V0complex of vacuolar-type H+-ATPases (V-ATPases). Mutations in the V-ATPasea3isoform have recently been shown to result in osteopetrosis, a fatal disease in humans, but no function has yet been ascribed to other isoforms. InCaenorhabditis elegans, theunc-32mutant was originally isolated on the basis of its movement defect. We have isolated four new mutant alleles, the strongest of which is embryonic lethal. We show here thatunc-32corresponds to one of the four genes encoding a V-ATPaseasubunit in the nematode, and we present their expression patterns and a molecular analysis of the gene family.unc-32gives rise via alternative splicing to at least six transcripts. In the uncoordinated alleles, the transcriptunc-32B is affected, suggesting that it encodes an isoform that is targeted to synaptic vesicles of cholinergic neurons, where it would control neurotransmitter uptake or release. Other isoforms expressed widely during embryogenesis are mutated in the lethal alleles and would be involved in other acidic organelles. Our results indicate that V-ATPaseasubunit genes are highly regulated and have tissue-specific function.

scholarly journals The ALBA RNA-binding proteins function redundantly to promote growth and flowering in Arabidopsis

2019 ◽  
Author(s):  
Naiqi Wang ◽  
Meachery Jalajakumari ◽  
Thomas Miller ◽  
Mohsen Asadi ◽  
Anthony A Millar
Keyword(s):  
Gene Expression ◽  
Protein Function ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Short Form ◽  
Expression Patterns ◽  
Wild Type ◽  
Long Form ◽  
Mrna Binding

AbstractRNA-binding proteins (RBPs) are critical regulators of gene expression, but have been poorly studied relative to other classes of gene regulators. Recently, mRNA-interactome capture identified many Arabidopsis RBPs of unknown function, including a family of ALBA domain containing proteins. Arabidopsis has three short-form ALBA homologues (ALBA1-3) and three long-form ALBA homologues (ALBA4-6), both of which are conserved throughout the plant kingdom. Despite this ancient origin, ALBA-GUS translational fusions of ALBA1, ALBA2, ALBA4, and ALBA5 had indistinguishable expression patterns, all being preferentially expressed in young, rapidly dividing tissues. Likewise, all four ALBA proteins had indistinguishable ALBA-GFP subcellular localizations in roots, all being preferentially located to the cytoplasm, consistent with being mRNA-binding. Genetic analysis demonstrated redundancy within the long-form ALBA family members; in contrast to single alba mutants that all appeared wild-type, a triple alba456 mutant had slower rosette growth and a strong delay in flowering-time. RNA-sequencing found most differentially expressed genes in alba456 were related to metabolism, not development. Additionally, changes to the alba456 transcriptome were subtle, suggesting ALBA4-6 participates in a process that does not strongly affect transcriptome composition. Together, our findings demonstrate that ALBA protein function is highly redundant, and is essential for proper growth and flowering in Arabidopsis.HighlightThe RNA-binding ALBA proteins have indistinguishable expression patterns and subcellular localizations in Arabidopsis, acting redundantly to promote growth and flowering via a mechanism that does not strongly affect transcriptome composition.

284.Expression of HtrA1, 2 and 3 in human endometrial cancer

2004 ◽  
Vol 16 (9) ◽  
pp. 284
Author(s):  
M. A. Bowden ◽  
L. A. Di Nezza ◽  
T. Jobling ◽  
L. A. Salamonsen ◽  
G. Nie
Keyword(s):  
Endometrial Cancer ◽  
High Temperature ◽  
Serine Protease ◽  
Cancer Progression ◽  
Serine Proteases ◽  
Genomic Structure ◽  
Normal Endometrium ◽  
Tissue Samples ◽  
Subtraction Hybridization ◽  
Temperature Requirement

The mammalian HtrA family consists of serine proteases with distinct domains homologous to the bacterial high temperature requirement factor (HtrA). Three human HtrA members have been reported: HtrA1 (PRSS11 or L56), HtrA2 (OMI) and HtrA3 (PRSP). The function of HtrA1 is not well characterised, but it has been shown to be downregulated in malignant tissues (1–3) indicating that the downregulation of HtrA1 is associated with cancer progression. HtrA2 regulates apoptosis by interacting with X-linked inhibitors of apoptosis (XIAP) thus preventing the caspase-inhibitory function of XIAP (4). The function of newly identified HtrA3 is not known, however it shares a high degree of sequence and domain homologies with HtrA1 and may therefore share a functional similarity with HtrA1 (5). Endometrial cancer (EC) is a prevalent gynaecological cancer, commonly affecting women after menopause. In this study we examined the expression of HtrA1, 2 and 3 in EC. Reverse transcriptase-PCR (semi-quantitative) analysis showed decreased mRNA expression of both HtrA1 and HtrA3, but no significant change for HtrA2, in EC tissue samples compared to normal endometrium. We then determined the protein level of expression and the cellular localisation of all three HtrA members in EC progression using immunohistochemistry. HtrA1 and HtrA3 showed a similar pattern of expression and both decreased dramatically with the progression of cancer from grade 1 through to 3. Surprisingly, HtrA2 protein expression was also decreased with cancer progression, but the decline was not as dramatic as that for HtrA1 and HtrA3. Interestingly, considerably less staining was observed for all three HtrA proteins in grade 3 cancer tissues. These data suggest that decreased expression of HtrA proteins, particularly HtrA1 and HtrA3, is associated with the progression of endometrial cancer. (1) Nie, G., Hampton, A., Li, Y., Findlay, J., Salamonsen, L.A. (2003) Identification and cloning of two isoforms of human high-temperature requirement factor A3 (HtrA3), characterization of its genomic structure and comparison of its tissue distribution with HtrA1 and HtrA2. Biochem. J. 371, 39–48. (2) van Loo, G., van Gurp, M., Depuydt, B., Srinivasula, S.M., Rodriguez, I., Alnemri, E.S., Gevaert, K., Vandekerckhove, J., Declercq, W., Vandenabeele, P. (2002) The serine protease OMI/HtrA2 is released from mitochondria during apoptosis. OMI interacts with caspase-inhibitor XIAP and induces enhanced caspase activity. Cell Death Diff. 9, 20–26. (3) Chien, J., Staub, J., Hu, S., Erickson-Johnson, M.R., Couch, F.J., Smith, D.I., Crowl, R.M., Kaufmann, S., Shridhar, V. (2004) A candidate tumour supressor HtrA1 is down-regulated in ovarian cancer. Oncogene 23, 1636–1644. (4) Shridhar, V., Sen, A., Chien, J., Staub, J., Avula, R., Kovats, S., Lee, J., Lillie, J., Smith, D.I. (2002) Identification of underexpressed genes in early- and late-stage primary ovarian tumours by suppression subtraction hybridization. Cancer Res. 62, 262–270. (5) Baldi, A., De Luca, A., Morini, M., Battista, T., Felsani, A., Baldi, F., Catricala, C., Amantea, A., Noonan, D. M., Albini, A., Ciorgio, P., Lombardi, D., Paggi, M. G. (2002) The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells. Oncogene 21, 6684–6688.

scholarly journals Expression and Localization of the Serine Proteases High-Temperature Requirement Factor A1, Serine Protease 23, and Serine Protease 35 in the Mouse Ovary

Endocrinology ◽  
2008 ◽  
Vol 149 (10) ◽  
pp. 5070-5077 ◽  
Author(s):  
Patrik Wahlberg ◽  
Åsa Nylander ◽  
Nina Ahlskog ◽  
Kui Liu ◽  
Tor Ny
Keyword(s):  
High Temperature ◽  
Serine Protease ◽  
Granulosa Cells ◽  
Serine Proteases ◽  
Expression Patterns ◽  
Follicular Development ◽  
Ovarian Stroma ◽  
Temperature Requirement ◽  
Extracellular Matrix Components ◽  
Preovulatory Follicles

Proteolytic degradation of extracellular matrix components has been suggested to play an essential role in the occurrence of ovulation. Recent studies in our laboratory have indicated that the plasminogen activator and matrix metalloproteinase systems, which were previously believed to be crucial for ovulation, are not required in this process. In this study we have used a microarray approach to identify new proteases that are involved in ovulation. We found three serine proteases that were relatively highly expressed during ovulation: high-temperature requirement factor A1 (HtrA1), which was not regulated much during ovulation; serine protease 23 (PRSS23), which was down-regulated by gonadotropins; and serine protease 35 (PRSS35), which was up-regulated by gonadotropins. We have further investigated the expression patterns of these proteases during gonadotropin-induced ovulation in immature mice and in the corpus luteum (CL) of pseudopregnant mice. We found that HtrA1 was highly expressed in granulosa cells throughout follicular development and ovulation, as well as in the forming and regressing CL. PRSS23 was highly expressed in atretic follicles, and it was expressed in the ovarian stroma and theca tissues just before ovulation. PRSS35 was expressed in the theca layers of developing follicles. It was also highly induced in granulosa cells of preovulatory follicles. PRSS35 was also expressed in the forming and regressing CL. These data suggest that HtrA1 and PRSS35 may be involved in ovulation and CL formation and regression, and that PRSS23 may play a role in follicular atresia.

Role of Alternative Splicing in Generating Isoform Diversity Among Plasma Membrane Calcium Pumps

2001 ◽  
Vol 81 (1) ◽  
pp. 21-50 ◽  
Author(s):  
Emanuel E. Strehler ◽  
David A. Zacharias
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Alternative Splicing ◽  
Splice Variants ◽  
Expression Patterns ◽  
Pdz Domain ◽  
Genetically Engineered ◽  
Intracellular Loop ◽  
Differential Distribution ◽  
Calcium Pumps

Calcium pumps of the plasma membrane (also known as plasma membrane Ca2+-ATPases or PMCAs) are responsible for the expulsion of Ca2+ from the cytosol of all eukaryotic cells. Together with Na+/Ca2+ exchangers, they are the major plasma membrane transport system responsible for the long-term regulation of the resting intracellular Ca2+concentration. Like the Ca2+ pumps of the sarco/endoplasmic reticulum (SERCAs), which pump Ca2+ from the cytosol into the endoplasmic reticulum, the PMCAs belong to the family of P-type primary ion transport ATPases characterized by the formation of an aspartyl phosphate intermediate during the reaction cycle. Mammalian PMCAs are encoded by four separate genes, and additional isoform variants are generated via alternative RNA splicing of the primary gene transcripts. The expression of different PMCA isoforms and splice variants is regulated in a developmental, tissue- and cell type-specific manner, suggesting that these pumps are functionally adapted to the physiological needs of particular cells and tissues. PMCAs 1 and 4 are found in virtually all tissues in the adult, whereas PMCAs 2 and 3 are primarily expressed in excitable cells of the nervous system and muscles. During mouse embryonic development, PMCA1 is ubiquitously detected from the earliest time points, and all isoforms show spatially overlapping but distinct expression patterns with dynamic temporal changes occurring during late fetal development. Alternative splicing affects two major locations in the plasma membrane Ca2+ pump protein: the first intracellular loop and the COOH-terminal tail. These two regions correspond to major regulatory domains of the pumps. In the first cytosolic loop, the affected region is embedded between a putative G protein binding sequence and the site of phospholipid sensitivity, and in the COOH-terminal tail, splicing affects pump regulation by calmodulin, phosphorylation, and differential interaction with PDZ domain-containing anchoring and signaling proteins. Recent evidence demonstrating differential distribution, dynamic regulation of expression, and major functional differences between alternative splice variants suggests that these transporters play a more dynamic role than hitherto assumed in the spatial and temporal control of Ca2+ signaling. The identification of mice carrying PMCA mutations that lead to diseases such as hearing loss and ataxia, as well as the corresponding phenotypes of genetically engineered PMCA “knockout” mice further support the concept of specific, nonredundant roles for each Ca2+ pump isoform in cellular Ca2+ regulation.

scholarly journals Dissecting the role of interprotomer cooperativity in the activation of oligomeric high-temperature requirement A2 protein

2021 ◽  
Vol 118 (35) ◽  
pp. e2111257118
Author(s):  
Yuki Toyama ◽  
Robert W. Harkness ◽  
Lewis E. Kay
Keyword(s):  
High Temperature ◽  
Ligand Binding ◽  
Pdz Domain ◽  
Nmr Studies ◽  
Transverse Relaxation ◽  
Temperature Requirement ◽  
Biochemical Assays ◽  
Substrate Concentrations ◽  
Binding Substrate

The human high-temperature requirement A2 (HtrA2) mitochondrial protease is critical for cellular proteostasis, with mutations in this enzyme closely associated with the onset of neurodegenerative disorders. HtrA2 forms a homotrimeric structure, with each subunit composed of protease and PDZ (PSD-95, DLG, ZO-1) domains. Although we had previously shown that successive ligand binding occurs with increasing affinity, and it has been suggested that allostery plays a role in regulating catalysis, the molecular details of how this occurs have not been established. Here, we use cysteine-based chemistry to generate subunits in different conformational states along with a protomer mixing strategy, biochemical assays, and methyl-transverse relaxation optimized spectroscopy–based NMR studies to understand the role of interprotomer allostery in regulating HtrA2 function. We show that substrate binding to a PDZ domain of one protomer increases millisecond-to-microsecond timescale dynamics in neighboring subunits that prime them for binding substrate molecules. Only when all three PDZ-binding sites are substrate bound can the enzyme transition into an active conformation that involves significant structural rearrangements of the protease domains. Our results thus explain why when one (or more) of the protomers is fixed in a ligand-binding–incompetent conformation or contains the inactivating S276C mutation that is causative for a neurodegenerative phenotype in mouse models of Parkinson’s disease, transition to an active state cannot be formed. In this manner, wild-type HtrA2 is only active when substrate concentrations are high and therefore toxic and unregulated proteolysis of nonsubstrate proteins can be suppressed.

Plant responses to high temperature: a view from pre-mRNA alternative splicing

2021 ◽  
Vol 105 (6) ◽  
pp. 575-583
Author(s):  
Jingya Lin ◽  
Ziqiang Zhu
Keyword(s):  
Alternative Splicing ◽  
High Temperature ◽  
Plant Responses
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