Requirement for Serine-384 in Caspase-2 processing and activity

Abstract Caspase-2 is a unique and conservative cysteine protease which plays an important role in several cellular processes including apoptotic cell death. Although the molecular mechanisms of its activation remain largely unclear, a major role belongs to the architecture of the caspase-2 active center. We demonstrate that the substitution of the putative phosphorylation site of caspase-2, Serine-384 to Alanine, blocks caspase-2 processing and decreases its enzymatic activity. Strikingly, in silico analysis using molecular dynamics simulations has shown that Serine-384 is crucially involved in interactions within the caspase-2 active center. It stabilizes Arginine-378, which forms a crucial hydrogen bond with the aspartate residue of a substrate. Hence, Serine-384 is essential for supporting a proper architecture of the active center of caspase-2. Moreover, molecular modeling strongly proved steric inaccessibility of Ser-384 to be phosphorylated. Importantly, a multiple alignment has demonstrated that both Serine-384 and Arg-378 residues are highly conservative across all members of caspase family, which allows us to suggest that this diade is indispensable for caspase processing and activity. Spontaneous mutations in this diade might influence oncosuppressive function of caspases, in particular of caspase-2. Likewise, the mutation of Ser-384 is associated with the development of lung squamous cell carcinoma and adenocarcinoma. Taken together, we have uncovered a central feature of the caspase-2 activation mechanism which is crucial for the regulation of its signaling network.

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The Role of Caspase-2 in Regulating Cell Fate

Cells ◽

10.3390/cells9051259 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1259

Author(s):

Vasanthy Vigneswara ◽

Zubair Ahmed

Keyword(s):

Cell Fate ◽

Molecular Mechanisms ◽

Apoptotic Cell ◽

Comprehensive Overview ◽

Cellular Processes ◽

Evolutionarily Conserved ◽

Caspase Family ◽

A Cell ◽

Caspase 2 ◽

Cycle Regulation

Caspase-2 is the most evolutionarily conserved member of the mammalian caspase family and has been implicated in both apoptotic and non-apoptotic signaling pathways, including tumor suppression, cell cycle regulation, and DNA repair. A myriad of signaling molecules is associated with the tight regulation of caspase-2 to mediate multiple cellular processes far beyond apoptotic cell death. This review provides a comprehensive overview of the literature pertaining to possible sophisticated molecular mechanisms underlying the multifaceted process of caspase-2 activation and to highlight its interplay between factors that promote or suppress apoptosis in a complicated regulatory network that determines the fate of a cell from its birth and throughout its life.

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Molecular Mechanisms of Resistance in Testicular Germ Cell Tumors - clinical Implications

Current Cancer Drug Targets ◽

10.2174/1568009618666180102103959 ◽

2018 ◽

Vol 18 (10) ◽

pp. 967-978 ◽

Cited By ~ 8

Author(s):

Katarina Kalavska ◽

Vincenza Conteduca ◽

Ugo De Giorgi ◽

Michal Mego

Keyword(s):

Germ Cell ◽

Molecular Mechanisms ◽

Cisplatin Resistance ◽

Apoptotic Cell ◽

Germ Cell Tumors ◽

Treatment Resistance ◽

Experimental Models ◽

Testicular Germ Cell Tumors ◽

Testicular Germ Cell ◽

Repair Capacity

Testicular germ cell tumors (TGCTs) represent the most common malignancy in men aged 15-35. Due to these tumors’ biological and clinical characteristics, they can serve as an appropriate system for studying molecular mechanisms associated with cisplatin-based treatment resistance. This review describes treatment resistance from clinical and molecular viewpoints. Cisplatin resistance is determined by various biological mechanisms, including the modulation of the DNA repair capacity of cancer cells, alterations to apoptotic cell death pathways, deregulation of gene expression pathways, epigenetic alterations and insufficient DNA binding. Moreover, this review describes TGCTs as a model system that enables the study of the cellular features of cancer stem cells in metastatic process and describes experimental models that can be used to study treatment resistance in TGCTs. All of the abovementioned aspects may help to elucidate the molecular mechanisms underlying cisplatin resistance and may help to identify promising new therapeutic targets.

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Molecular mechanisms of metabotropic GABAB receptor function

Science Advances ◽

10.1126/sciadv.abg3362 ◽

2021 ◽

Vol 7 (22) ◽

pp. eabg3362

Author(s):

Hamidreza Shaye ◽

Benjamin Stauch ◽

Cornelius Gati ◽

Vadim Cherezov

Keyword(s):

G Protein ◽

Molecular Mechanisms ◽

Gabab Receptor ◽

Neurological Diseases ◽

Activation Mechanism ◽

Allosteric Modulators ◽

Inhibitory Neurotransmitter ◽

Therapeutic Drugs ◽

G Protein Coupled ◽

The Brain

Metabotropic γ-aminobutyric acid G protein–coupled receptors (GABAB) represent one of the two main types of inhibitory neurotransmitter receptors in the brain. These receptors act both pre- and postsynaptically by modulating the transmission of neuronal signals and are involved in a range of neurological diseases, from alcohol addiction to epilepsy. A series of recent cryo-EM studies revealed critical details of the activation mechanism of GABAB. Structures are now available for the receptor bound to ligands with different modes of action, including antagonists, agonists, and positive allosteric modulators, and captured in different conformational states from the inactive apo to the fully active state bound to a G protein. These discoveries provide comprehensive insights into the activation of the GABAB receptor, which not only broaden our understanding of its structure, pharmacology, and physiological effects but also will ultimately facilitate the discovery of new therapeutic drugs and neuromodulators.

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Molecular Characterization and Phylogenetic Analysis of MADS-Box Gene VroAGL11 Associated with Stenospermocarpic Seedlessness in Muscadine Grapes

Genes ◽

10.3390/genes12020232 ◽

2021 ◽

Vol 12 (2) ◽

pp. 232

Author(s):

M Atikur Rahman ◽

Subramani P Balasubramani ◽

Sheikh M Basha

Keyword(s):

Phylogenetic Analysis ◽

Phosphorylation Site ◽

Orthologous Gene ◽

Mads Box ◽

Vitis Species ◽

Vitis Rotundifolia ◽

Seed Content ◽

Putative Phosphorylation Site ◽

Mads Box Gene ◽

Muscadine Grapes

Reduced expression of MADS-box gene AGAMOUS-LIKE11 (VviAGL11) is responsible for stenospermocarpic seedlessness in bunch grapes. This study is aimed to characterize the VviAGL11 orthologous gene (VroAGL11) in native muscadine grapes (Vitis rotundifolia) at the molecular level and analyze its divergence from other plants. The VroAGL11 transcripts were found in all muscadine cultivars tested and highly expressed in berries while barely detectable in leaves. RT-PCR and sequencing of predicted ORFs from diverse grape species showed that AGL11 transcripts were conservatively spliced. The encoded VroAGL11 protein contains highly conserved MADS-MEF2-like domain, MADS domain, K box, putative phosphorylation site and two sumoylation motifs. The muscadine VroAGL11 proteins are almost identical (99%) to that of seeded bunch cultivar, Chardonnay, except in one amino acid (A79G), but differs from mutant protein of seedless bunch grape, Sultanina, in two amino acids, R197L and T210A. Phylogenetic analysis showed that AGL11 gene of muscadine and other Vitis species formed a separate clade than that of other eudicots and monocots. Muscadine grape cultivar “Jane Bell” containing the highest percentage of seed content in berry (7.2% of berry weight) had the highest VroAGL11 expression, but almost none to nominal expression in seedless cultivars Fry Seedless (muscadine) and Reliance Seedless (bunch). These findings suggest that VroAGL11 gene controls the seed morphogenesis in muscadine grapes like in bunch grape and can be manipulated to induce stenospermocarpic seedlessness using gene editing technology.

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Carfilzomib in Combination with Bortezomib Enhances Apoptotic Cell Death in B16-F1 Melanoma Cells

Biology ◽

10.3390/biology10020153 ◽

2021 ◽

Vol 10 (2) ◽

pp. 153

Author(s):

Min Seung Lee ◽

So Hyun Lim ◽

Ah-Ran Yu ◽

Chi Yeon Hwang ◽

Insug Kang ◽

...

Keyword(s):

Er Stress ◽

Dose Reduction ◽

Molecular Mechanisms ◽

Apoptotic Cell ◽

Proteasome Inhibitors ◽

Annexin V ◽

Fluorescein Isothiocyanate ◽

Melanoma Cells ◽

Eif2α Phosphorylation ◽

Pharmacological Interactions

Proteasome inhibitors, such as bortezomib (BZ) and carfilzomib (CFZ), have been suggested as treatments for various cancers. To utilize BZ and/or CFZ as effective therapeutics for treating melanoma, we studied their molecular mechanisms using B16-F1 melanoma cells. Flow cytometry of Annexin V-fluorescein isothiocyanate-labeled cells indicated apoptosis induction by treatment with BZ and CFZ. Apoptosis was evidenced by the activation of various caspases, including caspase 3, 8, 9, and 12. Treatment with BZ and CFZ induced endoplasmic reticulum (ER) stress, as indicated by an increase in eIF2α phosphorylation and the expression of ER stress-associated proteins, including GRP78, ATF6α, ATF4, XBP1, and CCAAT/enhancer-binding protein homologous protein. The effects of CFZ on ER stress and apoptosis were lower than that of BZ. Nevertheless, CFZ and BZ synergistically induced ER stress and apoptosis in B16-F1 cells. Furthermore, the combinational pharmacological interactions of BZ and CFZ against the growth of B16-F1 melanoma cells were assessed by calculating the combination index and dose-reduction index with the CompuSyn software. We found that the combination of CFZ and BZ at submaximal concentrations could obtain dose reduction by exerting synergistic inhibitory effects on cell growth. Moreover, this drug combination reduced tumor growth in C57BL/6 syngeneic mice. Taken together, these results suggest that CFZ in combination with BZ may be a beneficial and potential strategy for melanoma treatment.

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Integrative cBioPortal Analysis Revealed Molecular Mechanisms That Regulate EGFR-PI3K-AKT-mTOR Pathway in Diffuse Gliomas of the Brain

Cancers ◽

10.3390/cancers13133247 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3247

Author(s):

Petar Brlek ◽

Anja Kafka ◽

Anja Bukovac ◽

Nives Pećina-Šlaus

Keyword(s):

Large Scale ◽

Molecular Mechanisms ◽

In Silico Analysis ◽

Total Sample ◽

Mtor Signaling ◽

Biological Behavior ◽

Mrna Transcription ◽

Diffuse Gliomas ◽

New Treatment ◽

Using Data

Diffuse gliomas are a heterogeneous group of tumors with aggressive biological behavior and a lack of effective treatment methods. Despite new molecular findings, the differences between pathohistological types still require better understanding. In this in silico analysis, we investigated AKT1, AKT2, AKT3, CHUK, GSK3β, EGFR, PTEN, and PIK3AP1 as participants of EGFR-PI3K-AKT-mTOR signaling using data from the publicly available cBioPortal platform. Integrative large-scale analyses investigated changes in copy number aberrations (CNA), methylation, mRNA transcription and protein expression within 751 samples of diffuse astrocytomas, anaplastic astrocytomas and glioblastomas. The study showed a significant percentage of CNA in PTEN (76%), PIK3AP1 and CHUK (75% each), EGFR (74%), AKT2 (39%), AKT1 (32%), AKT3 (19%) and GSK3β (18%) in the total sample. Comprehensive statistical analyses show how genomics and epigenomics affect the expression of examined genes differently across various pathohistological types and grades, suggesting that genes AKT3, CHUK and PTEN behave like tumor suppressors, while AKT1, AKT2, EGFR, and PIK3AP1 show oncogenic behavior and are involved in enhanced activity of the EGFR-PI3K-AKT-mTOR signaling pathway. Our findings contribute to the knowledge of the molecular differences between pathohistological types and ultimately offer the possibility of new treatment targets and personalized therapies in patients with diffuse gliomas.

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CD200 is a novel p53-target gene involved in apoptosis-associated immune tolerance

Blood ◽

10.1182/blood-2003-09-3184 ◽

2004 ◽

Vol 103 (7) ◽

pp. 2691-2698 ◽

Cited By ~ 54

Author(s):

Michael D. Rosenblum ◽

Edit Olasz ◽

Jeffery E. Woodliff ◽

Bryon D. Johnson ◽

Marja C. Konkol ◽

...

Keyword(s):

Target Gene ◽

Molecular Mechanisms ◽

Apoptotic Cell ◽

Immune Reactivity ◽

Biochemical Processes ◽

The Face ◽

P53 Target Gene ◽

Self Antigens

Abstract During apoptotic cell death, biochemical processes modify self-proteins and create potential autoantigens. To maintain self-tolerance in the face of natural cell turnover, the immune system must prevent or control responses to apoptosis-associated autoantigens or risk autoimmunity. The molecular mechanisms governing this process remain largely unknown. Here, we show that expression of the immunoregulatory protein CD200 increases as murine dendritic cells (DCs) undergo apoptosis. We define CD200 as a p53-target gene and identify both p53- and caspase-dependent pathways that control CD200 expression during apoptosis. CD200 expression on apoptotic DCs diminishes proinflammatory cytokine production in response to self-antigens in vitro and is required for UVB-mediated tolerance to haptenated self-proteins in vivo. Up-regulation of CD200 may represent a novel mechanism, whereby immune reactivity to apoptosis-associated self-antigens is suppressed under steady state conditions. (Blood. 2004;103: 2691-2698)

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The Actin-Binding Protein UNC-115/abLIM Controls Formation of Lamellipodia and Filopodia and Neuronal Morphogenesis in Caenorhabditis elegans

Molecular and Cellular Biology ◽

10.1128/mcb.25.12.5158-5170.2005 ◽

2005 ◽

Vol 25 (12) ◽

pp. 5158-5170 ◽

Cited By ~ 23

Author(s):

Yieyie Yang ◽

Erik A. Lundquist

Keyword(s):

Caenorhabditis Elegans ◽

Molecular Mechanisms ◽

Binding Protein ◽

Phosphorylation Site ◽

Serine Phosphorylation ◽

Actin Binding ◽

Axon Pathfinding ◽

Neuronal Morphogenesis ◽

Actin Binding Protein ◽

C Elegans

ABSTRACT The roles of actin-binding proteins in development and morphogenesis are not well understood. The actin-binding protein UNC-115 has been implicated in cytoskeletal signaling downstream of Rac in Caenorhabditis elegans axon pathfinding, but the cellular role of UNC-115 in this process remains undefined. Here we report that UNC-115 overactivity in C. elegans neurons promotes the formation of neurites and lamellipodial and filopodial extensions similar to those induced by activated Rac and normally found in C. elegans growth cones. We show that UNC-115 activity in neuronal morphogenesis is enhanced by two molecular mechanisms: when ectopically driven to the plasma membrane by the myristoylation sequence of c-Src, and by mutation of a putative serine phosphorylation site in the actin-binding domain of UNC-115. In support of the hypothesis that UNC-115 modulates actin cytoskeletal organization, we show that UNC-115 activity in serum-starved NIH 3T3 fibroblasts results in the formation of lamellipodia and filopodia. We conclude that UNC-115 is a novel regulator of the formation of lamellipodia and filopodia in neurons, possibly in the growth cone during axon pathfinding.

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Characterization and Expression Analysis of Genes Encoding Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxylase Kinase of Lotus japonicus, a Model Legume

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.4.281 ◽

2003 ◽

Vol 16 (4) ◽

pp. 281-288 ◽

Cited By ~ 19

Author(s):

Tomomi Nakagawa ◽

Tomoko Izumi ◽

Mari Banba ◽

Yosuke Umehara ◽

Hiroshi Kouchi ◽

...

Keyword(s):

Phosphoenolpyruvate Carboxylase ◽

Root Nodules ◽

Expression Patterns ◽

Phosphorylation Site ◽

Lotus Japonicus ◽

Specific Protein ◽

Mrna Levels ◽

Model Legume ◽

Putative Phosphorylation Site

Phosphoenolpyruvate carboxylases (PEPCs), one form of which in each legume species plays a central role in the carbon metabolism in symbiotic root nodules, are activated through phosphorylation of a conserved residue by a specific protein kinase (PEPC-PK). We characterized the cDNAs for two PEPC isoforms of Lotus japonicus, an amide-translocating legume that forms determinate nodules. One gene encodes a nodule-enhanced form, which is more closely related to the PEPCs in amide-type indeterminate nodules than those in ureide-type determinate nodules. The other gene is expressed in shoots and roots at a low level. Both forms have the putative phosphorylation site, Ser11. We also isolated a cDNA and the corresponding genomic DNA for PEPC-PK of L. japonicus. The recombinant PEPC-PK protein expressed in Escherichia coli phosphorylated recombinant maize C4-form PEPC efficiently in vitro. The level of mRNA for PEPC-PK was high in root nodules, and those in shoots and roots were also significant. In situ hybridization revealed that the expression patterns of the transcripts for PEPC and PEPC-PK were similar in mature root nodules, but were different in emerging nodules. When L. japonicus seedlings were subjected to prolonged darkness and subsequent illumination, the activity of PEPC-PK and the mRNA levels of both PEPC and PEPC-PK in nodules decreased and then recovered, suggesting that they are regulated according to the amounts of photosynthates transported from shoots.

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Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

eLife ◽

10.7554/elife.02172 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 40

Author(s):

Hiroshi Yamaguchi ◽

Toshihiko Maruyama ◽

Yoshihiro Urade ◽

Shigekazu Nagata

Keyword(s):

Adenosine Receptor ◽

Molecular Mechanisms ◽

Apoptotic Cell ◽

Adenosine Monophosphate ◽

Receptor Activation ◽

Death Process ◽

Apoptotic Cells ◽

Dependent Manner ◽

A2a Adenosine Receptor ◽

Anti Inflammatory

Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a ‘calm down’ signal.

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