257 research outputs found
Molecular Interactions of Prodiginines with the BH3 Domain of Anti-Apoptotic Bcl-2 Family Members.
No full textProdigiosin and obatoclax, members of the prodiginines family, are small molecules with anti-cancer properties that are currently under preclinical and clinical trials. The molecular target(s) of these agents, however, is an open question. Combining experimental and computational techniques we find that prodigiosin binds to the BH3 domain in some BCL-2 protein families, which play an important role in the apoptotic programmed cell death. In particular, our results indicate a large affinity of prodigiosin for MCL-1, an anti-apoptotic member of the BCL-2 family. In melanoma cells, we demonstrate that prodigiosin activates the mitochondrial apoptotic pathway by disrupting MCL-1/BAK complexes. Computer simulations with the PELE software allow the description of the induced fit process, obtaining a detailed atomic view of the molecular interactions. These results provide new data to understand the mechanism of action of these molecules, and assist in the development of more specific inhibitors of anti-apoptotic BCL-2 proteins
Prevention of cytokine withdrawal-induced apoptosis by Mcl-1 requires interaction between Mcl-1 and Bim
No full textGrowth factor withdrawal from hemopoietic cells results in activation of the mitochondrial pathway of apoptosis. Members of the Bcl-2 family regulate this pathway, with anti-apoptotic members counteracting the effects of pro-apoptotic members. We investigated the effect on Mcl-1 function of mutation at a conserved threonine 163 residue (T163) in its proline, glutamate, serine, and threonine rich (PEST) region. Under normal growth conditions, Mcl-1 half-life increased with alteration of T163 to glutamic acid, but decreased with mutation to alanine. However, both T163 mutants exhibited greater pro-survival effects compared with the wild type, which can be explained by an increased stability of the T163A mutant in cytokine-starved conditions. Both the mutant forms exhibited prolonged binding to pro-apoptotic Bim in cytokine-deprived cells. The extent to which Mcl-1 mutants were able to exert their anti-apoptotic effects correlated with their ability to associate with Bim. We further observed that primary bone marrow derived macrophages survived following cytokine withdrawal as long as Bim and Mcl-1 remained associated. In our study, we were unable to detect a role for GSK-3-mediated regulation of Mcl-1 expression. Based on these results we propose that upon cytokine withdrawal, survival of hemopoietic cells depends on association between Mcl-1 and Bim. Furthermore, alteration of T163 of Mcl-1 may change the protein such that its association with Bim is affected, resulting in prolonged association and increased survival. Le retrait de facteurs de croissance du milieu de croissance des cellules hémopoïétiques résulte en l'activation de la voie mitochondriale de l'apoptose. Les membres de la famille Bcl-2 régulent cette voie, les membres anti-apoptotiques contrecarrant les effets des membres pro-apoptotiques. Nous avons examiné l'effet de la mutation d'un résidu thréonine conservé (T163), présent dans la région riche en résidus proline, glutamate, sérine et thréonine (PEST), sur la fonction de Mcl-1. Le remplacement de la T163 par un acide glutamique augmentait la demi-vie de Mcl-1 en conditions de croissance normale, alors que la mutation en alanine la diminuait. Cependant, les deux types de mutations de la T163 favorisaient la survie comparativement au type sauvage, ce qui pouvait s'expliquer par une stabilité accrue des protéines mutantes en conditions de privation en cytokines. Les deux formes mutantes se liaient plus longtemps à Bim, un effecteur pro-apoptotique, chez les cellules privées de cytokines. La mesure avec laquelle les protéines Mcl-1 mutantes étaient capables d'exercer leurs effets anti-apoptotiques étaient corrélés avec leur capacité de s'associer à Bim. Nous avons ensuite observé que les macrophages primaires dérivés de la moelle osseuse survivaient après le retrait des cytokines tant et aussi longtemps que Bim et Mcl-1 restaient associés. Dans notre étude, nous avons été incapables de détecter un rôle de la régulation de l'expression de Mcl-1 par GSK-3. Nous proposons à partir de ces résultats qu'à la suite du retrait des cytokines, la survie des cellules hémopoïtiques dépend de l'association de Mcl-1 à Bim. De plus, la modification de la T163 de Mcl-1 pouvait changer la protéine de telle sorte que son association à Bim était affectée, résultant en une association prolongée et en une survie accrue. [ABSTRACT FROM AUTHOR]Peer reviewedfinal article publishedapoptosisprotein half-lifephosphorylationprotein–protein interactio
Loss of MCL-1 leads to impaired autophagy and rapid development of heart failure
No full textMyeloid cell leukemia-1 (MCL-1) is an anti-apoptotic BCL-2 protein that is up-regulated in several human cancers. MCL-1 is also highly expressed in myocardium, but its function in myocytes has not been investigated. We generated inducible, cardiomyocyte-specific Mcl-1 knockout mice and found that ablation of Mcl-1 in the adult heart led to rapid cardiomyopathy and death. Although MCL-1 is known to inhibit apoptosis, this process was not activated in MCL-1-deficient hearts. Ultrastructural analysis revealed disorganized sarcomeres and swollen mitochondria in myocytes. Mitochondria isolated from MCL-1-deficient hearts exhibited reduced respiration and limited Ca²⁺ -mediated swelling, consistent with opening of the mitochondrial permeability transition pore (mPTP). Double-knockout mice lacking MCL-1 and cyclophilin D, an essential regulator of the mPTP, exhibited delayed progression to heart failure and extended survival. Autophagy is normally induced by myocardial stress, but induction of autophagy was impaired in MCL-1-deficient hearts. These data demonstrate that MCL-1 is essential for mitochondrial homeostasis and induction of autophagy in the heart. This study also raises concerns about potential cardiotoxicity for chemotherapeutics that target MCL-1.Journal ArticleFinal article publishe
MCL-1 localizes to sites of DNA damage and regulates DNA damage response
No full textMCL-1, a pro-survival member of the BCL-2 family, was previously shown to have functions in ATR-dependent Chk1 phosphorylation following DNA damage. To further delineate these functions, we explored possible differences in DNA damage response caused by lack of MCL-1 in mouse embryo fibroblasts (MEFs). As expected, Mcl-1(-/-) MEFs had delayed Chk1 phosphorylation following etoposide treatment, compared to wild type MEFs. However, their response to hydroxyurea, which causes a G(1)/S checkpoint response, was not significantly different. In addition, appearance of gamma-H2AX was delayed in the Mcl-1(-/-) MEFs treated with etoposide. We next investigated whether MCL-1 is present, together with other DNA damage response proteins, at the sites of DNA damage. Immunoprecipitation of etoposide-treated extracts with anti-MCL-1 antibody showed association of MCL-1 with gamma-H2AX as well as NBS1. Immunofluorescent staining for MCL-1 further showed increased co-staining of MCL-1 and NBS1 following DNA damage. By using a system that creates DNA double strand breaks at specific sites in the genome, we demonstrated that MCL-1 is recruited directly adjacent to the sites of damage. Finally, in a direct demonstration of the importance of MCL-1 in allowing proper repair of DNA damage, we found that treatment for two brief exposures to etoposide , followed by periods of recovery, which mimics the clinical situation of etoposide use, resulted in greater accumulation of chromosomal abnormalities in the MEFs that lacked MCL-1. Together, these data indicate an important role for MCL-1 in coordinating DNA damage mediated checkpoint response, and have broad implications for the importance of MCL-1 in maintenance of genome integrity.Peer reviewedfinal article publishedprotein complexchromosomesG2/McheckpointDNA repai
An essential role for MCL-1 in ATR-mediated CHK1 phosphorylation
No full textHere we report a novel role for myeloid cell leukemia 1 (Mcl-1), a Bcl-2 family member, in regulating phosphorylation and activation of DNA damage checkpoint kinase, Chk1. Increased expression of nuclear Mcl-1 and/or a previously reported short nuclear form of Mcl-1, snMcl-1, was observed in response to treatment with low concentrations of etoposide or low doses of UV irradiation. We showed that after etoposide treatment, Mcl-1 could coimmunoprecipitate with the regulatory kinase, Chk1. Chk1 is a known regulator of DNA damage response, and its phosphorylation is associated with activation of the kinase. Transient transfection with Mcl-1 resulted in an increase in the expression of phospho-Ser345 Chk1, in the absence of any evidence of DNA damage, and accumulation of cells in G2. Importantly, knockdown of Mcl-1 expression abolished Chk1 phosphorylation in response to DNA damage. Mcl-1 could induce Chk1 phosphorylation in ATM-negative (ataxia telangectasia mutated) cells, but this response was lost in ATR (AT mutated and Rad3 related)-defective cells. Low levels of UV treatment also caused transient increases in Mcl-1 levels and an ATR-dependent phosphorylation of Chk1. Together, our results strongly support an essential regulatory role for Mcl-1, perhaps acting as an adaptor protein, in controlling the ATR-mediated regulation of Chk1 phosphorylation.Peer reviewedfinal article publishe
A proteolytic fragment of Mcl-1 exhibits nuclear localization and regulates cell growth by interaction with Cdk1
No full textMcl-1 (myeloid cell leukaemia-1) is a Bcl-2 family member with short-term pro-survival functions but whose other functions, demonstrated by embryonic lethality of knockout mice, do not involve apoptosis. In the present study, we show a cell-cycle-regulatory role of Mcl-1 involving a shortened form of the Mcl-1 polypeptide, primarily localized to the nucleus, which we call snMcl-1. snMcl-1 interacts with the cell-cycle-regulatory protein Cdk1 (cyclin-dependent kinase 1; also known as cdc2) in the nucleus, and Cdk1 bound to snMcl-1 was found to have a lower kinase activity. The interaction with Cdk1 occurs in the absence of its cyclin partners and is enhanced on treatment of cells with G2/M blocking agents, but not by G1/S blocking. The snMcl-1 polypeptide is present during S and G2 phases and is negligible in G1. Overexpression of human Mcl-1 in a murine myeloid progenitor cell line resulted in a lower rate of proliferation. Furthermore, Mcl-1-overexpressing cells had lower total Cdk1 kinase activity compared with parental cells, in both anti-Cdk1 and anti-cyclin B1 immunoprecipitates. The latter results suggest that binding to snMcl-1 alters the ability of Cdk1 to bind its conventional partner, cyclin B1. Given the important role of Cdk1 in progression through G2 and M phases, it is probable that the inhibition of Cdk1 activity accounts for the inhibitory effect of Mcl-1 on cell growth.Peer reviewedfinal article publishedCdkproteolysisnuclear localizationmyeloid cell leukaemia-1 (Mcl-1)kinasecell cycl
Simultaneous ACL Reconstruction and MCL Reconstruction in Patients with High BMI using Modified Lind Technique
No full textIntroduction:
Multiligamentous knee injury (MLKI) is a difficult and devastating injury of the knee defined as tear/disruption (involving grade III) of at least 2 of the 4 major ligaments of the knee. Combined anterior cruciate ligament (ACL) and medial collateral ligament (MCL) injuries are the most common type of MLKI. MCL injuries are concurrent in 20–38% of ACL injuries and are common in sports activities that involve pivoting of the knee joint, forced hyperextension, and rapid deceleration. Many techniques have been described for superficial MCL (sMCL) reconstruction, with single-bundle and double-bundle techniques used for the associated posterior oblique ligament (POL) using both allografts and autografts. Among these, one of the most common techniques with a good outcome (keeping the semitendinosus tibial attachment intact) was described by Lind et al. Our technique for sMCL and POL reconstruction is a modification of the Lind technique. In this technique, the semitendinosus with its intact tibial attachment is rerouted anatomically in the tibial tunnel with an adjustable loop, and on the femoral side, an adjustable loop UltraButton is used with a 2-incision technique. The remaining graft is reattached to the posteromedial tibia as POL using an interference screw
Material and Methods:
We treated patients with chronic ACL injuries combined with grade III valgus laxity. A total of 5 patients met the inclusion criteria of the study, and there were no patients lost to follow-up. The mean age was 26.5 years with a standard deviation of 4.05 years. All surgeries were performed by a single experienced author, Dr RK, at our institution between September 2023 and May 2024. The mean time from injury to surgery was 2.5 months, and the duration of follow-up was 6 months. 3 patients were female and 2 were male patients.
Results:
Out of 5 patients who were treated, 2 were in the age group of 15–20 years and 3 were 20–30 years. 2 were male patients and 3 were females. Road traffic accidents accounted for 66% (3 cases) of the total cases as the most common mechanism of injury followed by sports injuries (34%, 2 cases). All 5 patients operated on with simultaneous ACL and MCL reconstruction (modified Lind technique) had excellent results based on the Lysholm scoring system. Comparative analysis was done between pre-surgery and post-surgery Lysholm scores and we found that there was a statistically significant difference between them with P < 0.001. A significant improvement in the International Knee Documentation Committee subjective score was detected at follow-up.
Conclusion:
In patients with high body mass index >25 kg/m2, chronic ACL-MCL (grade III) injuries, simultaneous ACL-MCL reconstruction with the modified Lind technique improves anterior, valgus, and rotatory stability of the knee and produces a good functional result
Structural analysis for glycolipid recognition by the C-type lectins Mincle and MCL
No full textMincle [macrophage inducible Ca2+-dependent (C-type) lectin; CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are receptors for the cord factor TDM (trehalose-6,6'-dimycolate), a unique glycolipid of mycobacterial cell-surface components, and activate immune cells to confer adjuvant activity. Although it is known that receptor-TDM interactions require both sugar and lipid moieties of TDM, the mechanisms of glycolipid recognition by Mincle and MCL remain unclear. We here report the crystal structures of Mincle, MCL, and the Mincle-citric acid complex. The structures revealed that these receptors are capable of interacting with sugar in a Ca2+-dependent manner, as observed in other C-type lectins. However, Mincle and MCL uniquely possess shallow hydrophobic regions found adjacent to their putative sugar binding sites, which reasonably locate for recognition of fatty acid moieties of glycolipids. Functional studies using mutant receptors as well as glycolipid ligands support this deduced binding mode. These results give insight into the molecular mechanism of glycolipid recognition through C-type lectin receptors, which may provide clues to rational design for effective adjuvants
The Role of Mcl-1 in Embryonic Neural Precursor Cell Apoptosis
No full textMyeloid cell leukemia-1 (Mcl-1), an anti-apoptotic Bcl-2 protein, regulates neural precursor cell (NPC) survival in both the developing and adult mammalian nervous system. It is unclear when during the neurogenic period Mcl-1 becomes necessary for NPC survival and whether Bax is the sole pro-apoptotic target of Mcl-1. To address these questions, we used the nervous system-specific Nestin-Cre Mcl-1 conditional knockout mouse line (Mcl-1 CKO) to assess the anti-apoptotic role of Mcl-1 in developmental neurogenesis. Loss of Mcl-1 resulted in a wave of apoptosis beginning in the brainstem and cervical spinal cord at embryonic day 9.5 (E9.5) and in the forebrain at E10.5. Apoptosis was first observed ventrally in each region and spread dorsally over time. Within the spinal cord, apoptosis also spread in a rostral to caudal direction following the path of differentiation. Breeding the Mcl-1 CKO mouse with the Bax null mouse rescued the majority of NPC from apoptosis except in the dorsomedial brainstem and ventral thoracic spinal cord where only 50% were rescued. This demonstrates that Mcl-1 promotes NPC survival primarily by inhibiting the activation of Bax, but that Bax is not the sole pro-apoptotic target of Mcl-1 during embryonic neurogenesis. Interestingly, although co-deletion of Bax rescued the majority of NPC apoptosis, it resulted in embryonic lethality at E13, whereas conditional deletion of both Mcl-1 and Bax rescued embryonic lethality. In summary, this study demonstrates the widespread dependency on Mcl-1 during nervous system development.Memorial University Open Access Author's Fun
Myeloid cell leukemia-1 (MCL-1) siRNA therapy showed cytotoxic effect on T cells acute lymphoblastic leukemia
No full textBackground: T-lineage acute lymphoblastic leukemia (T-ALL) is a malignant hyperplastic disease of the hematopoietic system. This tumor is the most common tumor in children and adolescents. Myeloid cell leukemia-1 (Mcl-1) is described as a prosurvival protein from the Bcl2 family. It is an important factor in routine cancer treatments. In fact, in different types of cancers, Mcl-1 downregulation can be a potential target. Objectives: The present study aims to evaluate the cytotoxic effect of MCL-1 siRNA in T-ALL cells. Methods: The present study evaluated the effects of Mcl-1 small interfering RNAs (siRNAs) on survival in Jurkat cells. Specific Mcl-1 siRNA was transfected and using quantitative real-time PCR, the relative expression of Mcl-1 mRNA was determined. Moreover, cell survival was determined using the colorimetric MTT assay. Results: The expression of mRNA reduced effectively in a dose-dependent manner at 48 hours after transfection with Mcl-1 siRNA. In addition, Mcl-1 siRNA treatment could significantly reduce tumor cell survival. Conclusions: Based on the results, downregulation of Mcl-1 by specific siRNAs in T-ALL cells can effectively reduce cell survival. Therefore, Mcl-1 siRNA may be a complementary agent along with standard methods in the treatment of T-ALL. © 2019, Author(s)
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