Papers by Carla Finkielstein
Disabled-2 (Dab2) is an adaptor protein that regulates the extent of platelet aggregation by two ... more Disabled-2 (Dab2) is an adaptor protein that regulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin α iib β 3 receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with the pro-aggregatory mediators, the integrin α iib β 3 receptor and sulfatides, blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for sulfatide binding. Molecular docking suggested that a hydrophilic region, primarily mediated by R42, is responsible for interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with acyl chains. furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The Dab2 SBP residues that interact with sulfatides resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms. Abbreviations CD Circular dichroism Dab2 Disabled-2 DPC Dodecylphosphocholine GST Glutathione S-transferase N-PTB N-terminus containing the phosphotyrosine-binding domain SBP Sulfatide-binding peptide SPR Surface plasmon resonance The adaptor protein Disabled-2 (Dab2) is a multimodular signaling molecule involved in a variety of cellular processes including protein trafficking, cell growth and differentiation, cell adhesion, and modulation of platelet aggregation 1. Two Dab orthologs, Dab1 and Dab2, are present in mammals. Dab1 is primarily expressed in the brain 2 , whereas Dab2 is ubiquitously expressed in different tissues 3,4. Dab2 expression levels have been reported to exhibit a significant effect on cancer initiation and progression. Indeed, Dab2 expression is lost in breast, ovarian, prostate 5 , bladder 6 , and colorectal cancer cells 7 , suggesting that Dab2 can act as a tumor suppressor 8 .
Journal of Immunology, May 1, 2022
Since the beginning of the SARS-CoV-2 pandemic in early 2020, it was apparent that one of the mos... more Since the beginning of the SARS-CoV-2 pandemic in early 2020, it was apparent that one of the most striking clinical features of the disease caused by the virus, COVID-19, was the lung damage that occurs. This lung damage is often the result of an immune response gone awry as the host attempts to clear the virus. This overactive immune response, the ‘cytokine storm,’ is responsible for much of the most severe cases of COVID-19 as well as the Multisystem Inflammatory Syndrome in pediatric patients. Since the start of the pandemic, a few key players have been identified in the progression of COVID-19 disease as well as the immune response to it. These include pro-inflammatory cytokines IL-6, TNF-alpha, and CXCL10. Many clinical reports have especially shown the importance of the chemokine CXCL10 as a prognostic biomarker with patients having higher levels of CXCL10 often having more severe outcomes. Recent studies have also shown an increase in CXCL10 and its associated pathways in animal models of COVID-19. Here we continue to provide evidence for the importance of the protein CXCL10 in SARS-CoV-2 immunity through whole genome transcriptomics studies in human and k18-hACE2 mice. Following these, further studies with a mouse-adapted SARS-CoV-2 virus and Cxcl10−/− mice also showed a decreased level of effectiveness in viral clearance in the absence of effective CXCL10. These data continue to show the importance of the chemokine CXCL10 in both disease severity and viral clearance of SARS-CoV-2 infection adding to the growing body of evidence related to SARS-CoV-2 pathobiology.
International Journal of Biological Macromolecules, Nov 1, 2022
Apoptosis, or programmed cell death, is an essential process in all multi-cellular organisms. It ... more Apoptosis, or programmed cell death, is an essential process in all multi-cellular organisms. It is indispensable to an organism’s survival, preventing the malicious propagation of DNA damage and pathogenic alterations, through the clean disposal of afflicted cells. The BAD/tBID/BAK pathway is a portion of the apoptosis molecular pathway, albeit an important pathway since it is known to be deregulated and lead to pathological ailments such as cancer. Using chemical kinetics the BAD/tBID/BAK signaling pathway is modeled as a set of (nonlinear) ordinary differential equations. A first-cut numerical analysis reveals a mechanism where BAD sensitizes a switch from tBID activation to BAK activation. The phosphorylation of BAD is shown to inhibit this sensitizing effect. All behaviors are supported by experimental data, thereby validating the model of the BAD/tBID/BAK pathway. Moreover, modeling the phosphorylation of BAD as one of two modes, some conflicting experimental data about BAD’s phosphorylation can be studied. Parameter values (in this case the kinetic rate constants) are prone to error or missing altogether. Chemical reaction network theory, however, provides a theoretical approach to complement the initial numerical analysis without having to specify rate constant values. We extend the global asymptotic stability and robustness results in [92] to include any complex-balanced mass-action network. This enables us to study the BAD/tBID/BAK signaling network by breaking it into two sub-networks: one with BAD and tBID, and the other with tBID and BAK. The complex-balanced BAD/tBID sub-network is shown to possess a unique steady state which is globally asymptotically stable. This verifies the simple and dynamically well-behaved exchange of BAD for Bcl-2 proteins which guard against tBID activation. The second sub-network, tBID/BAK, is formulated as a complex-balanced network with a perturbation representing the reaction of tBID catalyzing the activation of BAK. Our theoretical results produce a non-conservative, though statedependent, condition which can be used to prove global convergence to a neighborhood of the unperturbed steady state. We then illustrate the biological importance of the result for tBID/BAK subnetwork with an example design for a drug delivery system.
Molecules and Cells, Oct 14, 2010
The Toll-interacting protein (Tollip) is a negative regulator of the Toll-like receptor (TLR)-med... more The Toll-interacting protein (Tollip) is a negative regulator of the Toll-like receptor (TLR)-mediated inflammation response. Tollip is a modular protein that contains an Nterminal Tom1-binding domain (TBD), a central conserved domain 2 (C2), and a C-terminal coupling of ubiquitin to endoplasmic reticulum degradation (CUE) domain. Here, we report the sequence-specific backbone 1 H, 15 N, and 13 C assignments of the human Tollip CUE domain. The CUE domain was found to be a stable dimer as determined by size-exclusion chromatography and molecular crosslinking studies. Analysis of the backbone chemical shift data indicated that the CUE domain exhibits three helical elements corresponding to 52% of the protein backbone. Circular dichroism spectrum analysis confirmed the helical nature of this domain. Comparison of the location of these helical regions with those reported for yeast CUE domains suggest differences in length for all helical elements. We expect the structural analysis presented here will be the foundation for future studies on the biological significance of the Tollip CUE domain, its molecular interactions, and the mechanisms that modulate its function during the inflammatory response.
Biochimica Et Biophysica Acta - Biomembranes, Nov 1, 2011
Disabled-2 (Dab2) is an adaptor protein involved in several biological processes ranging from end... more Disabled-2 (Dab2) is an adaptor protein involved in several biological processes ranging from endocytosis to platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2) at the inner leaflet of the plasma membrane. As a result of platelet activation, Dab2 is released from α-granules and associates with both the αIIbβ3 integrin receptor and sulfatide lipids on the platelet surface through its N-terminal region including the PTB domain (N-PTB), thus, modulating platelet aggregation. Thrombin, a strong platelet agonist, prevents Dab2 function by cleaving N-PTB within the two basic motifs required for sulfatide association, a reaction that is prevented when Dab2 is bound to these sphingolipids. We have characterized the membrane binding properties of Dab2 N-PTB using micelles enriched with Dab2 lipid ligands, sulfatides and PtdIns(4,5)P 2. Remarkably, NMR spectroscopy studies suggested differences in lipid-binding mechanisms. In addition, we experimentally demonstrated that sulfatide-and PtdIns(4,5)P 2-binding sites overlap in Dab2 N-PTB and that both lipids stabilize the protein against temperature-induced unfolding. We found that whereas sulfatides induced conformational changes and facilitated Dab2 N-PTB penetration into micelles, Dab2 N-PTB bound to PtdIns(4,5)P 2 lacked these properties. These results further support our model that platelet membrane sulfatides, but not PtdIns(4,5)P 2 , protect Dab2 N-PTB from thrombin cleavage.
Journal of Physical Chemistry B, Sep 27, 2010
Fifty percent of all cancer cases result from mutations of the TP53 gene, which encodes the tumor... more Fifty percent of all cancer cases result from mutations of the TP53 gene, which encodes the tumor suppressor p53, and it is hypothesized that the p53-mediated checkpoint pathway is compromised in most of the remaining cases. The p53 C-terminal domain (CTD) is an important site of p53 regulation but by nature is difficult to study, as it is intrinsically disordered. In this study, we performed molecular dynamics simulations on the p53 CTD and five known regulatory binding partners. We identified distinct trends in fluctuation within and around the p53 CTD binding site on each partner demonstrating a behavior that facilitates association. Further, we present evidence that the size of the hydrophobic pocket in each p53 CTD binding site governs the secondary structure of the p53 CTD when in the bound state. This information will be useful for predicting new binding partners for the p53 CTD, identifying interacting regions within other known partners, and discovering inhibitors that provide additional points of control over p53 activity.
PLOS ONE, Nov 24, 2009
Background: Platelets contact each other at the site of vascular injury to stop bleeding. One neg... more Background: Platelets contact each other at the site of vascular injury to stop bleeding. One negative regulator of platelet aggregation is Disabled-2 (Dab2), which is released to the extracellular surface upon platelet activation. Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB) domain by competing with fibrinogen for aIIbb3 integrin receptor binding by an unknown mechanism. Methodology/Principal Findings: Using protein-lipid overlay and liposome-binding assays, we identified that the Nterminal region of Dab2, including its PTB domain (N-PTB), specifically interacts with sulfatides. Moreover, we determined that such interaction is mediated by two conserved basic motifs with a dissociation constant (K d) of 0.6 mM as estimated by surface plasmon resonance (SPR) analysis. In addition, liposome-binding assays combined with mass spectroscopy studies revealed that thrombin, a strong platelet agonist, cleaved N-PTB at a site located between the basic motifs, a region that becomes protected from thrombin cleavage when bound to sulfatides. Sulfatides on the platelet surface interact with coagulation proteins, playing a major role in haemostasis. Our results show that sulfatides recruit N-PTB to the platelet surface, sequestering it from integrin receptor binding during platelet activation. This is a transient recruitment that follows N-PTB internalization by an actin-dependent process. Conclusions/Significance: Our experimental data support a model where two pools of Dab2 co-exist at the platelet surface, in both sulfatide-and integrin receptor-bound states, and their balance controls the extent of the clotting response.
BioEssays, Jul 1, 2016
Disabled-2 (Dab2) is a multimodular scaffold protein with signaling roles in the domains of cell ... more Disabled-2 (Dab2) is a multimodular scaffold protein with signaling roles in the domains of cell growth, trafficking, differentiation, and homeostasis. Emerging evidences place Dab2 as a novel modulator of cell-cell interaction; however, its mode of action has remained largely elusive. In this review, we highlight the relevance of Dab2 function in cell signaling and development and provide the most recent and comprehensive analysis of Dab2's action as a mediator of homotypical and heterotypical interactions. Accordingly, Dab-2 controls the extent of platelet aggregation through various motifs within its N-terminus. Dab2 interacts with the cytosolic tail of the integrin receptor blocking inside-out signaling, whereas extracellular Dab2 competes with fibrinogen for integrin α IIb β 3 receptor binding and, thus, modulates outside-in signaling. An additional level of regulation results from Dab2's association with cell surface lipids, an event that defines the extent of cell-cell interactions. As a multifaceted regulator, Dab2 acts as a mediator of endocytosis through its association with the [FY]xNPx[YF] motifs of internalized cell surface receptors, phosphoinositides, and clathrin. Other emerging roles of Dab2 include its participation in developmental mechanisms required for tissue formation and in modulation of immune responses. This review highlights the various novel mechanisms by which Dab2 mediates an array of signaling events with vast physiological consequences.
Journal of Biological Chemistry, Sep 1, 2004
The large T (LT) antigen encoded by SV40 virus is a multi-domain, multi-functional protein that c... more The large T (LT) antigen encoded by SV40 virus is a multi-domain, multi-functional protein that can not only transform cells but can also function as an efficient molecular machine to unwind duplex DNA for DNA replication. Here we report our findings on the oligomeric forms, domain interactions, and ATPase and helicase activities of various LT constructs. For the LT constructs that hexamerize, only two oligomeric forms, hexameric and monomeric, were detected in the absence of ATP/ADP. However, the presence of ATP/ADP stabilizes LT in the hexameric form. The LT constructs lacking the N-and C-terminal domains, but still retaining hexamerization ability, have ATPase as well as helicase activities at a level comparable to the full-length LT, suggesting the importance of hexamerization for these activities. The domain structures and the possible interactions between different LT fragments were probed with limited protease (trypsin) digestion. Such protease digestion generated a distinct pattern in the presence and absence of ATP/ADP and Mg 2؉. The most C-terminal fragment (residues 628-708, containing the host-range domain), which was thought to be completely unstructured, was somewhat trypsin-resistant despite the presence of multiple Arg and Lys, possibly due to a rather structured C terminus. Furthermore, the N-and Cterminal fragments cleaved by trypsin were associated with other parts of the molecule, suggesting the interdomain interactions for the fragments at both ends.
Molecular Pharmaceutics, Mar 16, 2017
We report the preparation of S-aroylthiooxime (SATO) functionalized amphiphilic block copolymer m... more We report the preparation of S-aroylthiooxime (SATO) functionalized amphiphilic block copolymer micelles that release hydrogen sulfide (H 2 S), a gaseous signaling molecule of relevance to various physiological and pathological conditions. The micelles release H 2 S in response to cysteine with a half-life of 3.3 h, which is substantially slower than a related small molecule SATO. Exogenous administration of H 2 S impacts growth and proliferation of cancer cells; however, the limited control over H 2 S generation from inorganic sulfide sources results in conflicting reports. Therefore, we compare the cellular cytotoxicity of SATO-functionalized micelles, which release H 2 S in a sustained manner, to Na 2 S, which releases H 2 S in a single dose. Our results show that H 2 S-releasing micelles significantly reduce the survival of HCT116 colon cancer cells relative to Na 2 S, GYY4137, and a small molecule SATO, indicating that release kinetics may play an important role in determining toxicity of H 2 S toward cancer cells. Furthermore, H 2 Sreleasing micelles are well tolerated by immortalized fibroblasts (NIH/3T3 cells), suggesting a selective toxicity of H 2 S toward cancer cells.
Cell, Oct 1, 2004
in vitro DNA replication. The OBD recognizes the viral origin of replication (Arthur et al., 1988... more in vitro DNA replication. The OBD recognizes the viral origin of replication (Arthur et al., 1988), which is required for the recruitment of LTag to the origin for DNA replication. To initiate replication, LTag first assembles at the ori
Night shift-work and circadian dysfunction have been linked to increased incidence of breast canc... more Night shift-work and circadian dysfunction have been linked to increased incidence of breast cancer. Circadian modulation of gene expression is often mediated by microRNAs (miRNAs), small 18-25 nucleotide noncoding RNA molecules that regulate posttranscriptional gene expression via mRNA inactivation. Although miRNA dysregulation has been implicated in breast cancer development, the relationship between breast cancer incidence in women exposed to chronic circadian disruption and their miRNA profiles has not been examined due to the lack of existing normal profiles of expression. We first identified miRNA expressed in asymptomatic breast tissue and defined their baseline profiles in women exposed to a regular work schedule compared to those who experienced chronic circadian disruption by night shift work. Normal mammary tissue was obtained from women undergoing therapeutic breast reductions. Clinical metadata on work, stress, sleep, pregnancy, medical, medication, family, and surgical history were obtained through survey and chart review. We selected night shift work starting from 0700 to 0300 as a marker of circadian disruption. Our studies focused on two major comparisons. First, we determined qualitative oscillatory differences in miRNA variants expressed during day and night. Second, we determined statistically significant expression levels of those day and night miRNAs in shift workers. Mammary miRNA reads were mapped against the reference human genome and mature miRNA set. One-way ANOVA was used to compare miRNA expression levels between night shift and non-night shift workers, as well as miRNA expression between other clinical metadata. At present, twelve samples have undergone experimental and statistical analyses. Three out of the twelve samples were from night shift workers. One-way ANOVA was run on the top one hundred expressed miRNAs including hsa-miR-143-3p, hsa-miR-99a-5p, hsa-miR-21-5p, hsa-miR-10b-5p, and hsa-miR-148a-3p to exclude miRNAs with zero or extremely low counts. Analysis showed no significant difference in miRNA expression of normal samples between night shift and non-night-shift workers (p 0.06099), pregnant and nulliparous (p 0.2658), and women who breastfed and those who did not (p 0.111). There was significant difference in miRNA expression of normal samples between patients who had breast cancer in the opposite breast compared to those who did not (p 0.03269) and patients with sleep problems and those who did not (p 0.003448). It is interesting to note that night shift work and sleep problems both represent forms of circadian disruption, yet only the latter showed significant difference in miRNA expression between controls. It is expected that patients with cancer in the other breast may exhibit significantly different reads from patients who have never had breast cancer. Future analyses will be performed using additional samples to build on these findings. Citation Format: Andrew Y. Li, Kristin Knight, Margaret Kneifel, Mark Feldmann, Kurtis Moyer, Allan Dickerman, Carla Finkielstein. Molecular profiling and threshold expression of microRNAs in mammary tissue of asymptomatic women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4446. doi:10.1158/1538-7445.AM2017-4446
The human Period 2 (hPer2) factor is a transcriptional regulator placed at the core of the circad... more The human Period 2 (hPer2) factor is a transcriptional regulator placed at the core of the circadian clock mechanism responsible for generating the negative feedback loop that sustains the clock. Its relevance to human diseases is underlined by alterations in its function that impacts many biochemical and physiological processes and, when absent, results in the development of various cancers. The tumor suppressor role of Per2 is speculated to involve transcriptional activation of p53, altered expression of cell cycle components, and regulation of the DNA-damage response pathway. However, it is entirely unclear how Per2 operates mechanistically. First, we identified hPer2 binds the C-terminus half of human p53 (hp53) and forms a stable trimeric complex with hp53’s negative regulator, the oncogenic protein Mdm2. Second, we determined that hPer2 binding to hp53 prevents Mdm2 from ubiquitinating and targeting hp53 by the proteasome. Accordingly, downregulation of hPer2 expression directly impacts hp53 levels whereas its overexpression influences both hp53 protein stability and transcription. Furthermore, we spatially define the distribution of the trimeric complex and determine the site for processing to be located in the nucleus. Third, we establish that hp53-mediated gene transcription is influenced by the presence of hPer2. Target genes such as 14-3-3σ, hp21WAF1/CIP1, and gadd45α show a synergistic increase in expression when hPer2 and hp53 are co-expressed in a hp53-deficient background. This result is the direct consequence of hPer2 dissociation of hp53 as result of checkpoint activation as shown by studies performed using a constitutively bound form of the hPer2/hp53 complex. Overall, our findings directly place hPer2 at the heart of the hp53-mediated response by modulating its stability and controlling its function. Citation Format: Carla V. Finkielstein, Tetsuya Gotoh, Marian Vila-Caballer, carlo santos, jingjing liu, jianhua yang. The circadian factor period 2 modulates p53 stability and function in downstream signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5173. doi:10.1158/1538-7445.AM2013-5173 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Research Square (Research Square), Jul 29, 2022
Reverse genetics systems are a critical tool in combating emerging viruses by enabling a better u... more Reverse genetics systems are a critical tool in combating emerging viruses by enabling a better understanding of the genetic mechanisms by which they cause disease. Traditional cloning approaches are fraught with di culties due to the bacterial toxicity of many viral sequences. Many attempts have been made to develop a simpli ed work ow to produce a simple-to-use infectious clone without the concerns of host toxicity; however, none offers the advantages of bacterial cloning without the various disadvantages. This paper demonstrates a novel in vitro work ow that leverages gene synthesis and replication cycle reaction-an innovative technology that reconstitutes the bacterial replication machinery in a tube-to develop a supercoiled infectious clone plasmid that is easy to rescue. Using this work ow, we developed two infectious clones, one of a low passage dengue virus serotype 2 isolate (PUO-218) and the Washington strain of SARS-CoV-2, which behaved similarly to their respective parental viruses. Furthermore, we generated a medically relevant mutant of SARS-CoV-2, Spike D614G. These results indicate that this novel in vitro work ow is a viable method to generate and manipulate infectious clones, speci cally for viruses that are notoriously di cult for traditional bacterial-based cloning methods. This work represents a paradigm shift in producing infectious clones and lowers the barrier of entry for scientists to develop such tools.
Journal of Molecular Endocrinology, Aug 1, 2016
In addition to the well-known function of ACTH as the main regulator of adrenal steroidogenesis, ... more In addition to the well-known function of ACTH as the main regulator of adrenal steroidogenesis, we have previously demonstrated its effect on the transcriptional stimulation of HO-1 expression, a component of the cellular antioxidant defense system. In agreement, we hereby demonstrate that, in adrenocortical Y1 cells, HO-1 induction correlates with a significant prevention of the generation of reactive oxygen species induced by H 2 O 2 /Fe 2+. ACTH/cAMP-dependent activation of redox-imbalanced related factors such as NRF2 or NFκB and the participation of MAPKs in this mechanism was, however, discarded based on results with specific inhibitors and reporter plasmids. We suggest the involvement of CREB in HO-1 induction by ACTH/cAMP, as transfection of cells with a dominant-negative isoform of CREB (DN-CREB-M1) decreased, while overexpression of CREB increased HO-1 protein levels. Sequence screening of the murine HO-1 promoter revealed CRE-like sites located at −146 and −37 of the transcription start site and ChIP studies indicated that this region recruits phosphorylated CREB (pCREB) upon cAMP stimulation in Y1 cells. In agreement, H89 (PKA inhibitor) or cotransfection with DN-CREB-M1 prevented the 8Br-cAMP-dependent increase in luciferase activity in cells transfected with pHO-1[−295/+74].LUC. ACTH and cAMP treatment induced the activation of the PI3K/Akt signaling pathway in a PKA-independent mechanism. Inhibition of this pathway prevented the cAMP-dependent increase in HO-1 protein levels and luciferase activity in cells transfected with pHO-1[−295/+74].LUC. Finally, here we show a crosstalk between the cAMP/PKA and PI3K pathways that affects the binding of p-CREB to its cognate element in the murine promoter of the Hmox1 gene.
BMC Developmental Biology, Oct 25, 2007
Background: The cell cycles of the Xenopus laevis embryo undergo extensive remodeling beginning a... more Background: The cell cycles of the Xenopus laevis embryo undergo extensive remodeling beginning at the midblastula transition (MBT) of early development. Cell divisions 2-12 consist of rapid cleavages without gap phases or cell cycle checkpoints. Some remodeling events depend upon a critical nucleo-cytoplasmic ratio, whereas others rely on a maternal timer controlled by cyclin E/ Cdk2 activity. One key event that occurs at the MBT is the degradation of maternal Wee1, a negative regulator of cyclin-dependent kinase (Cdk) activity. Results: In order to assess the effect of Wee1 on embryonic cell cycle remodeling, Wee1 mRNA was injected into one-cell stage embryos. Overexpression of Wee1 caused cell cycle delay and tyrosine phosphorylation of Cdks prior to the MBT. Furthermore, overexpression of Wee1 disrupted key developmental events that normally occur at the MBT such as the degradation of Cdc25A, cyclin E, and Wee1. Overexpression of Wee1 also resulted in post-MBT apoptosis, tyrosine phosphorylation of Cdks and persistence of cyclin E/Cdk2 activity. To determine whether Cdk2 was required specifically for the survival of the embryo, the cyclin E/Cdk2 inhibitor, ∆34-Xic1, was injected in embryos and also shown to induce apoptosis. Conclusion: Taken together, these data suggest that Wee1 triggers apoptosis through the disruption of the cyclin E/Cdk2 timer. In contrast to Wee1 and ∆34-Xic1, altering Cdks by expression of Chk1 and Chk2 kinases blocks rather than promotes apoptosis and causes premature degradation of Cdc25A. Collectively, these data implicate Cdc25A as a key player in the developmentally regulated program of apoptosis in X. laevis embryos.
Molecular Biology of the Cell, Oct 1, 2014
Human Period 2 (hPer2) is a transcriptional regulator at the core of the circadian clock mechanis... more Human Period 2 (hPer2) is a transcriptional regulator at the core of the circadian clock mechanism that is responsible for generating the negative feedback loop that sustains the clock. Its relevance to human disease is underlined by alterations in its function that affect numerous biochemical and physiological processes. When absent, it results in the development of various cancers and an increase in the cell's susceptibility to genotoxic stress. Thus we sought to define a yet-uncharacterized checkpoint node in which circadian components integrate environmental stress signals to the DNA-damage response. We found that hPer2 binds the C-terminal half of human p53 (hp53) and forms a stable trimeric complex with hp53's negative regulator, Mdm2. We determined that hPer2 binding to hp53 prevents Mdm2 from being ubiquitinated and targeting hp53 by the proteasome. Down-regulation of hPer2 expression directly affects hp53 levels, whereas its overexpression influences both hp53 protein stability and transcription of targeted genes. Overall our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to precondition the cell when a rapid, hp53-mediated, transcriptional response is needed.
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Papers by Carla Finkielstein