KCNN4 links PIEZO-dependent mechanotransduction to NLRP3 inflammasome activation.

Immune cells sense the microenvironment to fine-tune their inflammatory responses. Patients with cryopyrin-associated periodic syndrome (CAPS), caused by mutations in the NLRP3 gene, develop autoinflammation triggered by nonantigenic cues such as from the environment. However, the underlying mechanisms are poorly understood. Here, we uncover that KCNN4, a calcium-activated potassium channel, links PIEZO-mediated mechanotransduction to NLRP3 inflammasome activation. Yoda1, a PIEZO1 agonist, lowered the threshold for NLRP3 inflammasome activation. PIEZO-mediated sensing of stiffness and shear stress increased NLRP3-dependent inflammation. Myeloid-specific deletion of PIEZO1/2 protected mice from gouty arthritis. Mechanistically, activation of PIEZO1 triggers calcium influx, which activates KCNN4 to evoke potassium efflux and promotes NLRP3 inflammasome activation. Activation of PIEZO signaling was sufficient to activate the inflammasome in cells expressing CAPS-causing NLRP3 mutants via KCNN4. Last, pharmacological inhibition of KCNN4 alleviated autoinflammation in cells of patients with CAPS and in mice bearing a CAPS mutation. Thus, PIEZO-dependent mechanical inputs boost inflammation in NLRP3-dependent diseases, including CAPS.

Ubiquitin Binding Protein 2-Like (UBAP2L): is it so NICE After All?

Ubiquitin Binding Protein 2-like (UBAP2L, also known as NICE-4) is a ubiquitin- and RNA-binding protein, highly conserved in metazoans. Despite its abundance, its functions have only recently started to be characterized. Several studies have demonstrated the crucial involvement of UBAP2L in various cellular processes such as cell cycle regulation, stem cell activity and stress-response signaling. In addition, UBAP2L has recently emerged as a master regulator of growth and proliferation in several human cancers, where it is suggested to display oncogenic properties. Given that this versatile protein is involved in the regulation of multiple and distinct cellular pathways, actively contributing to the maintenance of cell homeostasis and survival, UBAP2L might represent a good candidate for future therapeutic studies. In this review, we discuss the current knowledge and latest advances on elucidating UBAP2L cellular functions, with an aim to highlight the importance of targeting UBAP2L for future therapies.

The NANOTUMOR consortium - Towards the Tumor Cell Atlas.

Cancer is a multi-step disease where an initial tumour progresses through critical steps shaping, in most cases, life-threatening secondary foci called metastases. The oncogenic cascade involves genetic, epigenetic, signalling pathways, intracellular trafficking and/or metabolic alterations within cancer cells. In addition, pre-malignant and malignant cells orchestrate complex and dynamic interactions with non-malignant cells and acellular matricial components or secreted factors within the tumour microenvironment that is instrumental in the progression of the disease. As our aptitude to effectively treat cancer mostly depends on our ability to decipher, properly diagnose and impede cancer progression and metastasis formation, full characterisation of molecular complexes and cellular processes at play along the metastasis cascade is crucial. For many years, the scientific community lacked adapted imaging and molecular technologies to accurately dissect, at the highest resolution possible, tumour and stromal cells behaviour within their natural microenvironment. In that context, the NANOTUMOR consortium is a French national multi-disciplinary workforce which aims at a providing a multi-scale characterisation of the oncogenic cascade, from the atomic level to the dynamic organisation of the cell in response to genetic mutations, environmental changes or epigenetic modifications. Ultimately, this program aims at identifying new therapeutic targets using innovative drug design.

UBASH3B-mediated silencing of the mitotic checkpoint: Therapeutic perspectives in cancer.

Defects in mitosis can lead to aneuploidy, which is a common feature of human cancers. Spindle Assembly Checkpoint (SAC) controls fidelity of chromosome segregation in mitosis to prevent aneuploidy. The ubiquitin receptor protein Ubiquitin Associated and SH3 Domain Containing B (UBASH3B) was recently found to control SAC silencing and faithful chromosome segregation by relocalizing Aurora B kinase to the mitotic microtubules. Accordingly, loss and gain of function of UBASH3B have strong effects on mitotic progression. Downregulation of UBASH3B prevents SAC satisfaction leading to inhibition of chromosome segregation, mitotic arrest, and cell death. In contrast, increased cellular levels of UBASH3B trigger premature and uncontrolled chromosome segregation. Interestingly, elevated levels of UBASH3B were found in aggressive tumors. Therefore, we raised the question whether the oncogenic potential of UBASH3B is linked to its role in chromosome segregation. Here we show that in cancer cells expressing high levels of UBASH3B and SAC proteins, downregulation of UBASH3B, can further potentiate SAC response inducing mitotic arrest and cell death. Moreover, data mining approaches identified a correlation between mRNA levels of UBASH3B and SAC components in a set of primary patient tumors including kidney and liver carcinomas. Thus, inhibition of UBASH3B may offer an attractive therapeutic perspective for cancers.

Mutations in the HECT domain of NEDD4L lead to AKT-mTOR pathway deregulation and cause periventricular nodular heterotopia.

Neurodevelopmental disorders with periventricular nodular heterotopia (PNH) are etiologically heterogeneous, and their genetic causes remain in many cases unknown. Here we show that missense mutations in NEDD4L mapping to the HECT domain of the encoded E3 ubiquitin ligase lead to PNH associated with toe syndactyly, cleft palate and neurodevelopmental delay. Cellular and expression data showed sensitivity of PNH-associated mutants to proteasome degradation. Moreover, an in utero electroporation approach showed that PNH-related mutants and excess wild-type NEDD4L affect neurogenesis, neuronal positioning and terminal translocation. Further investigations, including rapamycin-based experiments, found differential deregulation of pathways involved. Excess wild-type NEDD4L leads to disruption of Dab1 and mTORC1 pathways, while PNH-related mutations are associated with deregulation of mTORC1 and AKT activities. Altogether, these data provide insights into the critical role of NEDD4L in the regulation of mTOR pathways and their contributions in cortical development.

Molecular dynamics of PLK1 during mitosis.

Polo-like kinase 1 (PLK1) is a key regulator of eukaryotic cell division. During mitosis, dynamic regulation of PLK1 is crucial for its roles in centrosome maturation, spindle assembly, microtubule-kinetochore attachment, and cytokinesis. Similar to other members of the PLK family, the molecular architecture of PLK1 protein is characterized by 2 domains-the kinase domain and the regulatory substrate-binding domain (polo-box domain)-that cooperate and control PLK1 function during mitosis. Mitotic cells employ many layers of regulation to activate and target PLK1 to different cellular structures in a timely manner. During the last decade, numerous studies have shed light on the precise molecular mechanisms orchestrating the mitotic activity of PLK1 in time and space. This review aims to discuss available data and concepts related to regulation of the molecular dynamics of human PLK1 during mitotic progression.

CUL3 and protein kinases: insights from PLK1/KLHL22 interaction.

Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22. (1) In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases.

The emerging family of CULLIN3-RING ubiquitin ligases (CRL3s): cellular functions and disease implications.

Protein ubiquitylation is a post-translational modification that controls all aspects of eukaryotic cell functionality, and its defective regulation is manifested in various human diseases. The ubiquitylation process requires a set of enzymes, of which the ubiquitin ligases (E3s) are the substrate recognition components. Modular CULLIN-RING ubiquitin ligases (CRLs) are the most prevalent class of E3s, comprising hundreds of distinct CRL complexes with the potential to recruit as many and even more protein substrates. Best understood at both structural and functional levels are CRL1 or SCF (SKP1/CUL1/F-box protein) complexes, representing the founding member of this class of multimeric E3s. Another CRL subfamily, called CRL3, is composed of the molecular scaffold CULLIN3 and the RING protein RBX1, in combination with one of numerous BTB domain proteins acting as substrate adaptors. Recent work has firmly established CRL3s as major regulators of different cellular and developmental processes as well as stress responses in both metazoans and higher plants. In humans, functional alterations of CRL3s have been associated with various pathologies, including metabolic disorders, muscle, and nerve degeneration, as well as cancer. In this review, we summarize recent discoveries on the function of CRL3s in both metazoans and plants, and discuss their mode of regulation and specificities.

Ubiquitylation-dependent localization of PLK1 in mitosis.

Polo-like kinase 1 (PLK1) critically regulates mitosis through its dynamic localization to kinetochores, centrosomes and the midzone. The polo-box domain (PBD) and activity of PLK1 mediate its recruitment to mitotic structures, but the mechanisms regulating PLK1 dynamics remain poorly understood. Here, we identify PLK1 as a target of the cullin 3 (CUL3)-based E3 ubiquitin ligase, containing the BTB adaptor KLHL22, which regulates chromosome alignment and PLK1 kinetochore localization but not PLK1 stability. In the absence of KLHL22, PLK1 accumulates on kinetochores, resulting in activation of the spindle assembly checkpoint (SAC). CUL3-KLHL22 ubiquitylates Lys 492, located within the PBD, leading to PLK1 dissociation from kinetochore phosphoreceptors. Expression of a non-ubiquitylatable PLK1-K492R mutant phenocopies inactivation of CUL3-KLHL22. KLHL22 associates with the mitotic spindle and its interaction with PLK1 increases on chromosome bi-orientation. Our data suggest that CUL3-KLHL22-mediated ubiquitylation signals degradation-independent removal of PLK1 from kinetochores and SAC satisfaction, which are required for faithful mitosis.

Decoding ubiquitin for mitosis.

Conjugation of ubiquitin (ubiquitination) to substrate proteins is a widespread modification that ensures fidelity of many cellular processes. During mitosis, different dynamic morphological transitions have to be coordinated in a temporal and spatial manner to allow for precise partitioning of the genetic material into two daughter cells, and ubiquitination of key mitotic factors is believed to provide both directionality and fidelity to this process. While directionality can be achieved by a proteolytic type of ubiquitination signal, the fidelity is often determined by various types of ubiquitin conjugation that does not target substrates for proteolysis by the proteasome. An additional level of complexity is provided by various ubiquitin-interacting proteins that act downstream of the ubiquitinated substrate and can serve as "decoders" for the ubiquitin signal. They may, specifically reverse ubiquitin attachment (deubiquitinating enzymes, DUBs) or, act as a receptor for transfer of the ubiquitinated substrate toward downstream signaling components and/or subcellular compartments (ubiquitin-binding proteins, UBPs). In this review, we aim at summarizing the knowledge and emerging concepts about the role of ubiquitin decoders, DUBs, and UBPs that contribute to faithful regulation of mitotic division.

A Cul3-based E3 ligase regulates mitosis and is required to maintain the spindle assembly checkpoint in human cells.

The spindle assembly checkpoint (SAC) is a mechanism that prevents premature chromosome segregation in anaphase before all chromosomes are correctly attached to the mitotic spindle. Errors in chromosome segregation lead to aneuploidy, which may be causally involved in tumorgenesis. Kinetochore complexes are the structural components of the SAC, which are tightly regulated by various mechanisms including phosphorylation and ubiquitin-dependent proteolysis. Recent studies shed new light on the regulatory pathways of the ubiquitin proteasome system involved in SAC signaling. Here we present evidence that a Cul3-based E3 ubiquitin-ligase is required to maintain SAC signaling in human cells. Inactivation of the Cul3/KLHL9/KLHL13 ligase leads to premature degradation of Cyclin B and exit from the mitotic state in the presence of microtubule poisons. We discuss possible mechanisms how Cul3 may be required to maintain SAC activity by ubiquitination of the chromosomal passenger protein Aurora B.

c-Jun/AP-1 controls liver regeneration by repressing p53/p21 and p38 MAPK activity.

The AP-1 transcription factor c-Jun is a key regulator of hepatocyte proliferation. Mice lacking c-Jun in the liver (c-jun (Deltali*)) display impaired liver regeneration after partial hepatectomy (PH). This phenotype correlates with increased protein levels of the cdk-inhibitor p21 in the liver. We performed PH experiments in several double-knockout mouse models to genetically identify the signaling events regulated by c-Jun. Inactivation of p53 in c-jun (Deltali*) mice abrogated both hepatocyte cell cycle block and increased p21 protein expression. Consistently, liver regeneration was rescued in c-jun (Deltali*) p21 (-/-) double-mutant mice. This indicated that c-Jun controls hepatocyte proliferation by a p53/p21-dependent mechanism. Analyses of p21 mRNA and protein expression in livers of c-jun (Deltali*) mice after PH revealed that the accumulation of p21 protein is due to a post-transcriptional/post-translational mechanism. We have investigated several candidate pathways implicated in the regulation of p21 expression, and observed increased activity of the stress kinase p38 in regenerating livers of c-jun (Deltali*) mice. Importantly, conditional deletion of p38alpha in livers of c-jun (Deltali*) mice fully restored hepatocyte proliferation and attenuated increased p21 protein levels after PH. These data demonstrate that c-Jun/AP-1 regulates liver regeneration through a novel molecular pathway that involves p53, p21, and the stress kinase p38alpha.

Distinct functions of junD in cardiac hypertrophy and heart failure.

Cardiac hypertrophic stimuli induce both adaptive and maladaptive growth response pathways in heart. Here we show that mice lacking junD develop less adaptive hypertrophy in heart after mechanical pressure overload, while cardiomyocyte-specific expression of junD in mice results in spontaneous ventricular dilation and decreased contractility. In contrast, fra-1 conditional knock-out mice have a normal hypertrophic response, whereas hearts from fra-1 transgenic mice decompensate prematurely. Moreover, fra-1 transgenic mice simultaneously lacking junD reveal a spontaneous dilated cardiomyopathy associated with increased cardiomyocyte apoptosis and a primary mitochondrial defect. These data suggest that junD promotes both adaptive-protective and maladaptive hypertrophy in heart, depending on its expression levels.

Requirement of JNK2 for scavenger receptor A-mediated foam cell formation in atherogenesis.

In vitro studies suggest a role for c-Jun N-terminal kinases (JNKs) in proatherogenic cellular processes. We show that atherosclerosis-prone ApoE-/- mice simultaneously lacking JNK2 (ApoE-/- JNK2-/- mice), but not ApoE-/- JNK1-/- mice, developed less atherosclerosis than do ApoE-/- mice. Pharmacological inhibition of JNK activity efficiently reduced plaque formation. Macrophages lacking JNK2 displayed suppressed foam cell formation caused by defective uptake and degradation of modified lipoproteins and showed increased amounts of the modified lipoprotein-binding and -internalizing scavenger receptor A (SR-A), whose phosphorylation was markedly decreased. Macrophage-restricted deletion of JNK2 was sufficient to decrease atherogenesis. Thus, JNK2-dependent phosphorylation of SR-A promotes uptake of lipids in macrophages, thereby regulating foam cell formation, a critical step in atherogenesis.

Regulation of sister chromatid cohesion between chromosome arms.

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.

The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase.

The separation of sister chromatids in anaphase depends on the dissociation of cohesin from chromosomes. In vertebrates, some cohesin is removed from chromosomes at the onset of anaphase by proteolytic cleavage. In contrast, the bulk of cohesin is removed from chromosomes already in prophase and prometaphase by an unknown mechanism that does not involve cohesin cleavage. We show that Polo-like kinase is required for the cleavage-independent dissociation of cohesin from chromosomes in Xenopus. Cohesin phosphorylation depends on Polo-like kinase and reduces the ability of cohesin to bind to chromatin. These results suggest that Polo-like kinase regulates the dissociation of cohesin from chromosomes early in mitosis.