Multimeric Complexes Research Articles

Exploitation of ATP-sensitive potassium ion (KATP) channels by HPV promotes cervical cancer cell proliferation by contributing to MAPK/AP-1 signalling.

Persistent infection with high-risk human papillomaviruses (HPVs) is the causal factor in multiple human malignancies, including >99% of cervical cancers and a growing proportion of oropharyngeal cancers. Prolonged expression of the viral oncoproteins E6 and E7 is necessary for transformation to occur. Although some of the mechanisms by which these oncoproteins contribute to carcinogenesis are well-characterised, a comprehensive understanding of the signalling pathways manipulated by HPV is lacking. Here, we present the first evidence to our knowledge that the targeting of a host ion channel by HPV can contribute to cervical carcinogenesis. Through the use of pharmacological activators and inhibitors of ATP-sensitive potassium ion (KATP) channels, we demonstrate that these channels are active in HPV-positive cells and that this activity is required for HPV oncoprotein expression. Further, expression of SUR1, which forms the regulatory subunit of the multimeric channel complex, was found to be upregulated in both HPV+ cervical cancer cells and in samples from patients with cervical disease, in a manner dependent on the E7 oncoprotein. Importantly, knockdown of SUR1 expression or KATP channel inhibition significantly impeded cell proliferation via induction of a G1 cell cycle phase arrest. This was confirmed both in vitro and in in vivo tumourigenicity assays. Mechanistically, we propose that the pro-proliferative effect of KATP channels is mediated via the activation of a MAPK/AP-1 signalling axis. A complete characterisation of the role of KATP channels in HPV-associated cancer is now warranted in order to determine whether the licensed and clinically available inhibitors of these channels could constitute a potential novel therapy in the treatment of HPV-driven cervical cancer.

ASH2L regulates postnatal neurogenesis through Onecut2-mediated inhibition of TGF-β signaling pathway.

The ability of neural stem/progenitor cells (NSPCs) to proliferate and differentiate is required through different stages of neurogenesis. Disturbance in the regulation of neurogenesis causes many neurological diseases, such as intellectual disability, autism, and schizophrenia. However, the intrinsic mechanisms of this regulation in neurogenesis remain poorly understood. Here, we report that Ash2l (Absent, small or homeotic discs-like 2), one core component of a multimeric histone methyltransferase complex, is essential for NSPC fate determination during postnatal neurogenesis. Deletion of Ash2l in NSPCs impairs their capacity for proliferation and differentiation, leading to simplified dendritic arbors in adult-born hippocampal neurons and deficits in cognitive abilities. RNA sequencing data reveal that Ash2l primarily regulates cell fate specification and neuron commitment. Furthermore, we identified Onecut2, a major downstream target of ASH2L characterized by bivalent histone modifications, and demonstrated that constitutive expression of Onecut2 restores defective proliferation and differentiation of NSPCs in adult Ash2l-deficient mice. Importantly, we identified that Onecut2 modulates TGF-β signaling in NSPCs and that treatment with a TGF-β inhibitor rectifies the phenotype of Ash2l-deficient NSPCs. Collectively, our findings reveal the ASH2L-Onecut2-TGF-β signaling axis that mediates postnatal neurogenesis to maintain proper forebrain function.

Evaluation of AlphaFold-Multimer prediction on multi-chain protein complexes.

Despite near-experimental accuracy on single-chain predictions, there is still scope for improvement among multimeric predictions. Methods like AlphaFold-Multimer and FoldDock can accurately model dimers. However, how well these methods fare on larger complexes is still unclear. Further, evaluation methods of the quality of multimeric complexes are not well established. We analysed the performance of AlphaFold-Multimer on a homology-reduced dataset of homo- and heteromeric protein complexes. We highlight the differences between the pairwise and multi-interface evaluation of chains within a multimer. We describe why certain complexes perform well on one metric (e.g. TM-score) but poorly on another (e.g. DockQ). We propose a new score, Predicted DockQ version 2 (pDockQ2), to estimate the quality of each interface in a multimer. Finally, we modelled protein complexes (from CORUM) and identified two highly confident structures that do not have sequence homology to any existing structures. All scripts, models, and data used to perform the analysis in this study are freely available at https://gitlab.com/ElofssonLab/afm-benchmark.

P-027 Absence of perinuclear theca ACTRT1 protein induces sperm head deformation and primary male infertility in humans

Abstract Study question Given that Actrt1 knockout induces teratospermia, fertilization failure and severe male infertility in mice, will ACTRT1 deficiency affect sperm morphology and male fertility in humans? Summary answer The ∼110 kb microdeletion of X chromosome only including ACTRT1 gene was identified inducing sperm head deformation and fertilization failure in an infertile Chinese man. What is known already The actin-related proteins, ACTRT1, ACTRT2, ACTL7A, and ACTL9, interact to form a multimeric complex in the sperm subacrosomal region and is vital for the attachment of acrosome to nucleus. The disruption of ACTL7A or ACTL9 have been identified to cause acrosomal detachment, total fertilization failure and male infertility in humans and mice. Actrt1-knockout mice are severely subfertile also because of malformed sperm heads with detached acrosomes and a partial deficiency in fertilization. Loss/reduced expression and/or abnormal localization of PLCζ, a well-known sperm-borne activating factor, are identified in ACTL7A/ACTL9 mutant patients as well as Actrt1-, Actl7a-, and Actl9-deficient mice. Study design, size, duration We recruited a cohort of 85 infertile men with teratospermia which is characterized by deformed sperm heads in the center for reproductive medicine from August 2019 to June 2022. Genomic DNA (gDNA) of patients was extracted from peripheral blood, then whole-exome sequencing and in silico analyses were performed to identify gene mutations. Morphological analysis, functional assays, and assisted reproductive therapy were performed in 2022. Participants/materials, setting, methods The ACTRT1 deficiency was identified by whole-exome sequencing and confirmed by whole-genome sequencing, PCR and qPCR. Family members’ gDNA was collected to define the hereditary mode. Papanicolaou-staining, scanning and transmission electronic microscopy were performed to reveal sperm morphologies. Western blot and immunostaining of spermatozoa were conducted to explore the pathological mechanism of the ACTRT1 deficiency. Intracytoplasmic sperm injection (ICSI) combined with artificial oocyte activation (AOA) was applied for the assisted reproductive therapy of ACTRT1-deletion patient. Main results and the role of chance We identified a whole-gene deletion mutation of ACTRT1 on Chromosome X in an infertile male with teratospermia, which was inherited from his mother. Papanicolaou-staining, scanning electronic microscopy, and transmission electronic microscopy showed the sperm head deformation owing to acrosome detachment, which mimicked the previously reported phenotype of Actrt1-knockout mice. The results of western blot and immunostaining suggested that ACTRT1 deficiency induced a down-regulated expression of ACTL7A and PLCζ proteins, but not ACTRT2, in human sperm samples. The detached acrosomes induced fertilization failure in assisted reproductive therapy of both Actrt1-knockout mice and the ACTRT1-deletion patient, which could be effectively rescued by ICSI combined with AOA. Limitations, reasons for caution Additional cases are needed to confirm the genetic contribution of ACTRT1 mutations to male infertility with teratospermia and fertilization failure. In addition, the proband with ACTRT1 deletion was with severe oligozoospermia and mild asthenozoospermia. The effect of ACTRT1 mutations on sperm count and motility still need more investigation. Wider implications of the findings Our results reveal a gene–disease relationship between ACTRT1 deficiency and human male infertility owing to teratospermia and fertilization failure. This report also describe a good fertility outcome of assisted reproductive therapy with ICSI and AOA for the ACTRT1-deficient patient. Trial registration number not applicable

MTORC1/rpS6 and mTORC2/PKC regulate spermatogenesis through Arp3-mediated actin microfilament organization in Eriocheir sinensis.

Mammalian target of rapamycin (mTOR) is a crucial signaling protein regulating a range of cellular events. Numerous studies have reported that the mTOR pathway is related to spermatogenesis in mammals. However, its functions and underlying mechanisms in crustaceans remain largely unknown. mTOR exists as two multimeric functional complexes termed mTOR complex 1 (mTORC1) and mTORC2. Herein, we first cloned ribosomal protein S6 (rpS6, a downstream molecule of mTORC1) and protein kinase C (PKC, a downstream effector of mTORC2) from the testis of Eriocheir sinensis. The dynamic localization of rpS6 and PKC suggested that both proteins may be essential for spermatogenesis. rpS6/PKC knockdown and Torin1 treatment led to defects in spermatogenesis, including germ cell loss, retention of mature sperm and empty lumen formation. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was disrupted in the rpS6/PKC knockdown and Torin1 treatment groups, accompanied by changing in expression and distribution of junction proteins. Further study demonstrated that these findings may result from the disorganization of filamentous actin (F-actin) networks, which were mediated by the expression of actin-related protein 3 (Arp3) rather than epidermal growth factor receptor pathway substrate 8 (Eps8). In summary, our study illustrated that mTORC1/rpS6 and mTORC2/PKC regulated spermatogenesis via Arp3-mediated actin microfilament organization in E. sinensis.

Mesenchymal stem cells (MSCs) and MSC-derived exosomes in animal models of central nervous system diseases: Targeting the NLRP3 inflammasome.

The NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome is a multimeric protein complex that is engaged in the innate immune system and plays a vital role in inflammatory reactions. Activation of the NLRP3 inflammasome and subsequent release of proinflammatory cytokines can be triggered by microbial infection or cellular injury. The NLRP3 inflammasome has been implicated in the pathogenesis of many disorders affecting the central nervous system (CNS), ranging from stroke, traumatic brain injury, and spinal cord injury to Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, and depression. Furthermore, emerging evidence has suggested that mesenchymal stem cells (MSCs) and their exosomes may modulate NLRP3 inflammasome activation in a way that might be promising for the therapeutic management of CNS diseases. In the present review, particular focus is placed on highlighting and discussing recent scientific evidence regarding the regulatory effects of MSC-based therapies on the NLRP3 inflammasome activation and their potential to counteract proinflammatory responses and pyroptotic cell death in the CNS, thereby achieving neuroprotective impacts and improvement in behavioral impairments.

BE3 is the major branching enzyme isoform required for amylopectin synthesis in Chlamydomonas reinhardtii.

Starch-branching enzymes (BEs) are essential for starch synthesis in both plants and algae where they influence the architecture and physical properties of starch granules. Within Embryophytes, BEs are classified as type 1 and type 2 depending on their substrate preference. In this article, we report the characterization of the three BE isoforms encoded in the genome of the starch producing green algae Chlamydomonas reinhardtii: two type 2 BEs (BE2 and BE3) and a single type 1 BE (BE1). Using single mutant strains, we analyzed the consequences of the lack of each isoform on both transitory and storage starches. The transferred glucan substrate and the chain length specificities of each isoform were also determined. We show that only BE2 and BE3 isoforms are involved in starch synthesis and that, although both isoforms possess similar enzymatic properties, BE3 is critical for both transitory and storage starch metabolism. Finally, we propose putative explanations for the strong phenotype differences evidenced between the C. reinhardtii be2 and be3 mutants, including functional redundancy, enzymatic regulation or alterations in the composition of multimeric enzyme complexes.

An Artemisia scoparia extract attenuates glucocorticoid-induced lipolysis in adipocytes.

Prescription glucocorticoid (GC) use is widespread across developed countries for the treatment of several inflammatory conditions. Elevated GCs are known to promote lipolysis and metabolic disorders. An extract of Artemisia scoparia (SCO) has been shown to reduce lipolysis and promote metabolic health but has not been investigated in the context of excess GCs. Our aim was to examine the effects of SCO on GC-induced lipolysis. Mature adipocytes were pretreated with vehicle or SCO, then exposed to either the synthetic GC dexamethasone (DEX) or tumor necrosis factor alpha (TNFα). Medium was collected and assayed for glycerol and fatty acids as measures of lipolysis. The expression of several lipolytic genes and proteins was assessed, and the involvement of glucocorticoid receptor (GR) in SCO's effects was also interrogated. SCO significantly attenuated DEX-induced lipolysis but did not interfere with DEX-mediated changes in inflammatory gene profiles in adipocytes. SCO treatment resulted in significant reductions in monomeric phosphodiesterase (PDE) protein levels while elevating PDE multimeric complex formation, but other canonical lipolytic mediators were unaltered. SCO attenuated lipolysis even when GR expression was significantly knocked down. Finally, it was demonstrated that SCO was distinct from rosiglitazone in its antilipolytic effects. SCO attenuates GC-induced lipolysis independently of GR activity. Future studies are needed to elucidate underlying mechanisms.

In the Rat Midbrain, SG2NA and DJ-1 have Common Interactome, Including Mitochondrial Electron Transporters that are Comodulated Under Oxidative Stress.

Scaffold proteins Striatin and SG2NA assemble kinases and phosphatases into the signalling complexes called STRIPAK. Dysfunctional STRIPAKs cause cancer, cerebral cavernous malformations, etc. DJ-1, a sensor for oxidative stress, has long been associated with the Parkinson's disease, cancer, and immune disorders. SG2NA interacts with DJ-1 and Akt providing neuroprotection under oxidative stress. To dissect the role of SG2NA and DJ-1 in neuronal pathobiology, rat midbrain extracts were immunoprecipitated with SG2NA and sixty-three interacting proteins were identified. BN-PAGE followed by the LC-MS/MS showed 1030 comigrating proteins as the potential constituents of the multimeric complexes formed by SG2NA. Forty-three proteins were common between those identified by co-immunoprecipitation and the BN-PAGE. Co-immunoprecipitation with DJ-1 identified 179 interacting partners, of which forty-one also interact with SG2NA. Among those forty-one proteins immunoprecipitated with both SG2NA and DJ-1, thirty-nine comigrated with SG2NA in the BN-PAGE, and thus are bonafide constituents of the supramolecular assemblies comprising both DJ-1 and SG2NA. Among those thirty-nine proteins, seven are involved in mitochondrial oxidative phosphorylation. In rotenone-treated rats having Parkinson's like symptoms, the levels of both SG2NA and DJ-1 increased in the mitochondria; and the association of SG2NA with the electron transport complexes enhanced. In the hemi-Parkinson's model, where the rats were injected with 6-OHDA into the midbrain, the occupancy of SG2NA and DJ-1 in the mitochondrial complexes also increased. Our study thus reveals a new family of potential STRIPAK assemblies involving both SG2NA and DJ-1, with key roles in protecting midbrain from the oxidative stress.

Active Zone Trafficking of CaV2/UNC-2 Channels Is Independent of β/CCB-1 and α2δ/UNC-36 Subunits.

The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming α1 subunit and two auxiliary β and α2δ subunits. Although auxiliary subunits are critical for channel function, whether they are required for α1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans, we show that UNC-2/α1 localizes to AZs even in the absence of CCB-1/β or UNC-36/α2δ, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes.SIGNIFICANCE STATEMENT Synaptic transmission in the neuron hinges on the coupling of synaptic vesicle exocytosis with calcium influx. This calcium influx is mediated by CaV2 voltage-gated calcium channels. These channels consist of one pore-forming α1 subunit and two auxiliary β and α2δ subunits. The auxiliary subunits enhance channel function and regulate the overall level of channels at presynaptic terminals. However, it is not settled how these auxiliary subunits regulate the overall channel level. Our study in C. elegans finds that although the auxiliary subunits do not coassemble with α1 and aid trafficking, they are recruited to α1 and stabilize the channel complex at presynaptic terminals. Our study suggests that drugs that target the auxiliary subunits can directly destabilize and have an impact on CaV2 channels.

Hemato-vascular specification requires arnt1 and arnt2 genes in zebrafish embryos.

During embryonic development, a subset of cells in the mesoderm germ layer are specified as hemato-vascular progenitor cells, which then differentiate into endothelial cells and hematopoietic stem and progenitor cells. In zebrafish, the transcription factor npas4l (cloche) is required for the specification of hemato-vascular progenitor cells. However, it is unclear whether npas4l is the sole factor at the top of the hemato-vascular specification cascade. Here, we show that arnt1 and arnt2 genes are required for hemato-vascular specification. We found that arnt1;arnt2 double mutant zebrafish embryos, but not arnt1 or arnt2 single mutants, lack blood cells and most endothelial cells. arnt1/2 mutants have reduced or absent expression of etsrp and tal1, the earliest known endothelial and hematopoietic transcription factor genes. We found that Npas4l binds both Arnt1 and Arnt2 proteins in vitro, consistent with the idea that PAS domain-containing bHLH transcription factors act in a multimeric complex to regulate gene expression. Our results demonstrate that npas4l, arnt1 and arnt2 act together to regulate endothelial and hematopoietic cell fate, where each gene is necessary, but not sufficient, to drive hemato-vascular specification.

MTOR signaling in renal ion transport.

The mammalian target of rapamycin (mTOR) signaling pathway is crucial in maintaining cell growth and metabolism. The mTOR protein kinase constitutes the catalytic subunit of two multimeric protein complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). As such, this pathway is indispensable for many organs, including the kidney. Since its discovery, mTOR has been associated with major renal disorders such as acute kidney injury, chronic kidney disease, and polycystic kidney disease. On top of that, emerging studies using pharmacological interventions and genetic disease models have unveiled mTOR role in renal tubular ion handling. Along the tubule, mTORC1 and mTORC2 subunits are ubiquitously expressed at mRNA level. Nevertheless, at the protein level, current studies suggest that a tubular segment-specific balance between mTORC1 and mTORC2 exists. In the proximal tubule, mTORC1 regulates nutrients transports through various transporters located in this segment. On the other hand, in the thick ascending limb of the loop of Henle, both complexes play a role in regulating NKCC2 expression and activity. Lastly, in the principal cells of the collecting duct, mTORC2 determines Na+ reabsorption and K+ excretion by regulating of SGK1 activation. Altogether, these studies establish the relevance of the mTOR signaling pathway in the pathophysiology of tubular solute transport. Despite extensive studies on the effectors of mTOR, the upstream activators of mTOR signaling remain elusive in most nephron segments. Further understanding of the role of growth factor signaling and nutrient sensing is essential to establish the exact role of mTOR in kidney physiology.

Abstract 1435: A GABP dominant negative approach to targeting tumor cell immortality

Abstract Immortality is a hallmark of human cancer cells and therapeutic reversal is of great interest. Telomerase Reverse Transcriptase promoter (TERTp) mutations reactivate TERT expression, the rate limiting step in telomerase activity and cellular immortality. While prior telomerase blocking therapies lack tumor selectivity and were poorly tolerated, TERTp mutations and their regulation offer a unique opportunity for tumor specific reversal of cellular immortality. TERTp mutations are the most common non-coding mutation across all cancers, including glioblastoma (GBM), melanoma, urothelial carcinoma, and many others. The mutations generate a de novo E26 Transformation Specific (ETS) binding motif that recruits the GA-Binding Protein (GABP) multimeric complex to reactivate TERT expression. While GABP can form both a dimeric and tetrameric complex, we found that the tetrameric complex is responsible for reactivation of the mutant TERTp, suggesting selectively targeting the tetramer may induce telomere shortening and tumor cell death. However, through knocking out the tetramer, we found that the GABP dimer is sufficiently upregulated to maintain TERT expression. In the absence of the dimer and the tetramer isoform, many tumor cells senesce or undergo cell death. However, some escape this fate via upregulation of a second tetramer forming GABP paralogue that maintains TERT expression. Therefore, we devised a transactivation domain null GABP subunit that we hypothesized would act in a dominant negative manner to block the GABP dimer and both GABP tetramers. Introduction of the dominant negative construct into TERTp mutant GBM, meningioma, medulloblastoma, oligodendroglioma, and urothelial carcinoma cells significantly reduced TERT expression while not altering expression in TERTp wildtype GBM cells. Furthermore, the dominant negative is able to shorten telomere length in TERTp mutant GBM cells. While therapeutic targeting of the three GABP complexes seems particularly challenging, our initial dominant negative appears to do so. Ultimately the dominant negative could be delivered directly into tumors via clinically approved viral delivery systems such as retroviral replicating vectors. Citation Format: Nicholas O. Stevers, Sara A. Collins, Samuel H. Wu, Noriyuki Kasahara, Joseph F. Costello. A GABP dominant negative approach to targeting tumor cell immortality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1435.

Abstract 1474: Hippo-mediated control of the mutant TERT promoter via MYC and GABP

Abstract Most cancers achieve replicative immortality through reactivation of telomerase reverse transcriptase (TERT), the rate limiting subunit in telomerase activity. Tumors from more than 50 distinct cancer types reactivate TERT expression via mutations in the TERT promoter. Understanding how the mutations lead to activation and immortality is a central question in cancer research. Oncogenic signaling pathways including EGFR/AMPK, MAPK, and C-MYC/ASL, have been linked to telomere maintenance via their regulation of the mutant TERT promoter. Two mutually exclusive point mutations in the TERT promoter generate a de novo E26 Transformation Specific (ETS) binding motif that recruits the GA-Binding Protein (GABP) multimeric complex to reactivate TERT expression (Bell et al, Science 2015). Recruitment of GABP to the mutant TERT promoter facilitates the recruitment of C-MYC to the promoter (Shi et al, Mol. Cell 2022), which aids in transcriptional activation. The Hippo pathway plays a key role in development, organ size determination, and homeostasis, and has been found dysregulated in numerous cancer types. GABP is a member of the Hippo pathway, with GABP activating the promoter of the Hippo transcriptional effector Yes-associated protein (YAP1) and the Hippo pathway core kinases stimulating GABP nuclear exit (Wu et al., Cell Reports 2013). We have found that YAP and its paralogue transcriptional coactivator with PDZ-binding motif (TAZ) are linked to mutant TERT promoter activation in glioblastoma via transcriptional activation of both GABPA and MYC. These observations suggest the Hippo pathway supports tumor immortality in glioblastoma. While immortality has proven to be an elusive target, further investigations of the signaling networks that converge on the activation of the mutant TERT promoter may reveal therapeutic targets that block tumor growth and have secondary benefit by their impact on tumor immortality. Citation Format: Nicholas O. Stevers, Andrew Mancini, Joseph F. Costello. Hippo-mediated control of the mutant TERT promoter via MYC and GABP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1474.

Mechanoelectrical transduction-related genetic forms of hearing loss.

Hair cells of the mammalian cochlea are specialized mechanosensory cells that convert mechanical stimuli into electrical signals to initiate the neuronal responses that lead to the perception of sound. The mechanoelectrical transduction (MET) machinery of cochlear hair cells is a multimeric protein complex that consists of the pore forming subunits of the MET channel and several essential accessory subunits that are crucial to regulate channel function and render the channel mechanically sensitive. Mutations have been discovered in the genes that encode all known components of the MET machinery. These mutations cause hearing loss with or without vestibular dysfunction. Some mutations also affect other tissues such as the retina. In this brief review, we will summarize gene mutations that affect the MET machinery of hair cells and how the study of the affected genes has illuminated our understanding of the physiological role of the encoded proteins.

The MAX2-KAI2 module promotes salicylic acid-mediated immune responses in Arabidopsis.

Arabidopsis MORE AXILLARY GROWTH2 (MAX2) is a key component in the strigolactone (SL) and karrikin (KAR) signaling pathways and regulates the degradation of SUPPRESSOR OF MAX2 1/SMAX1-like (SMAX1/SMXL) proteins, which are transcriptional co-repressors that regulate plant architecture, as well as abiotic and biotic stress responses. The max2 mutation reduces resistance against Pseudomonas syringae pv. tomato (Pst). To uncover the mechanism of MAX2-mediated resistance, we evaluated the resistance of various SL and KAR signaling pathway mutants. The resistance of SL-deficient mutants and of dwarf 14 (d14) was similar to that of the wild-type, whereas the resistance of the karrikin insensitive 2 (kai2) mutant was compromised, demonstrating that the KAR signaling pathway, not the SL signaling pathway, positively regulates the immune response. We measured the resistance of smax1 and smxl mutants, as well as the double, triple, and quadruple mutants with max2, which revealed that both the smax1 mutant and smxl6/7/8 triple mutant rescue the low resistance phenotype of max2 and that SMAX1 accumulation diminishes resistance. The susceptibility of smax1D, containing a degradation-insensitive form of SMAX1, further confirmed the SMAX1 function in the resistance. The relationship between the accumulation of SMAX1/SMXLs and disease resistance suggested that the inhibitory activity of SMAX1 to resistance requires SMXL6/7/8. Moreover, the exogenous application of KAR2 enhanced resistance against Pst, but KAR-induced resistance depended on salicylic acid (SA) signaling. Inhibition of karrikin signaling delayed SA-mediated defense responses and inhibited pathogen-induced protein biosynthesis. Together, we propose that the MAX2-KAI2-SMAX1 complex regulates resistance with the assistance of SMXL6/7/8 and SA signaling and that SMAX1/SMXLs possibly form a multimeric complex with their target transcription factors to fine tune immune responses.

Behind Base J: The Roles of JBP1 and JBP2 on Trypanosomatids

β-D-glucopyranosyloxymethiluracil (Base J) is a modified thymidine base found in kinetoplastids and some related organisms. Interestingly, Base J distribution into the genome can vary depending on the organism and its life stage. Base J is reported to be found mostly at telomeric repeats, on inactive variant surface glycoproteins (VSG's) expression sites (e.g., T. brucei), in RNA polymerase II termination sites and sub-telomeric regions (e.g., Leishmania). This hypermodified nucleotide is synthesized in two steps with the participation of two distinct thymidine hydroxylases, J-binding protein 1 and 2 (JBP1 and JBP2, respectively) and a β-glucosyl transferase. A third J-binding protein, named JBP3, was recently identified as part of a multimeric complex. Although its structural similarities with JBP1, it seems not to be involved in J biosynthesis but to play roles in gene expression regulation in trypanosomatids. Over the years, with the characterization of JBP1 and JBP2 mutant lines, Base J functions have been targeted and shone a light on that matter, showing genus-specific features. This review aims to explore Base J's reported participation as a regulator of RNA polymerase II transcription termination and to summarize the functional and structural characteristics and similarities of the remarkable JBP proteins in pathogenic trypanosomatids.

Chronic activation of inflammasome signaling complexes and enhancement of behavioral abnormalities

IntroductionInflammasomes are cytosolic multi-component signaling platforms critical to the innate immune response to infectious diseases and the dysregulation of their activation can lead to the development of neurodegeneration and cancer.ObjectivesWe aim through this review to ass a possible interplay between dysregulation of inflammasome activation, development of chronic inflammatory disease and enhancement of behavioral abnormalities.MethodsWe comprehensively review the scientific literature using Pubmed database and other search platforms such as Google scholar to assess the role and the actors of chronic activation of inflammasome signaling complexes to establish a potential association between dysregulation of inflammasome activation, chronic inflammatory disease and enhancement of behavioral abnormalities.ResultsOur bibliographic review revealed that dysregulation of the inflammasome is associated with the onset and progression of several autoinflammatory and autoimmune diseases, including cryopyrin-associated periodic fever syndrome, familial Mediterranean fever, rheumatoid arthritis, and systemic lupus erythematosus. These multimeric complexes form in response to molecular patterns unique to pathogens and cellular damage, triggering a cascade of downstream responses, including the induction of pyroptotic cell death and release of proinflammatory cytokines. Some inflammasomes directly recognize these patterns, while others indirectly sense these patterns through changes in the homeostatic environment of the cell. Moreover, although being a normal part of the skin flora, yeasts of the genus Malassezia are associated with several inflammatory skin diseases including pityriasis versicolor (tinea versicolor), atopic eczema, psoriasis, Malassezia folliculitis and onychomycoses. In the context of tolerating fungi during colonization and eliciting, activation, of inflammasomes signaling complexes, has been identified as an integral part of antifungal host defense. While the activation of inflammasomes mainly the NLRP3 one, was shown to be pivotal for innate immunity against pathogenic fungi such as candida albicans, their role in the fungal genus Malassezia remains imprecise. Even though, many observations suggest that simultaneous activation of NLRP3, NLRC4 and AIM2 inflammasomes may play an important role.ConclusionsWhereas, chronic inflammasome activation such as by chronic infectious has been tied to the development of metabolic syndromes, neurodegenerative diseases, and cancer progression, a possible interplay between chronic invasion by the genus Malassezia, vigorous immune response to eliminate invading fungal pathogens, disruption of immune sensors of genotoxic stress, development of chronic inflammatory disease and behavioral abnormalities may be a new field of scientific researches.Disclosure of InterestNone Declared

SMN post-translational modifications in spinal muscular atrophy.

Since its first identification as the gene responsible for spinal muscular atrophy (SMA), the range of survival motor neuron (SMN) protein functions has increasingly expanded. This multimeric complex plays a crucial role in a variety of RNA processing pathways. While its most characterized function is in the biogenesis of ribonucleoproteins, several studies have highlighted the SMN complex as an important contributor to mRNA trafficking and translation, axonal transport, endocytosis, and mitochondria metabolism. All these multiple functions need to be selectively and finely modulated to maintain cellular homeostasis. SMN has distinct functional domains that play a crucial role in complex stability, function, and subcellular distribution. Many different processes were reported as modulators of the SMN complex activities, although their contribution to SMN biology still needs to be elucidated. Recent evidence has identified post-translational modifications (PTMs) as a way to regulate the pleiotropic functions of the SMN complex. These modifications include phosphorylation, methylation, ubiquitination, acetylation, sumoylation, and many other types. PTMs can broaden the range of protein functions by binding chemical moieties to specific amino acids, thus modulating several cellular processes. Here, we provide an overview of the main PTMs involved in the regulation of the SMN complex with a major focus on the functions that have been linked to SMA pathogenesis.

EIF4A1-dependent mRNAs employ purine-rich 5'UTR sequences to activate localised eIF4A1-unwinding through eIF4A1-multimerisation to facilitate translation.

Altered eIF4A1 activity promotes translation of highly structured, eIF4A1-dependent oncogene mRNAs at root of oncogenic translational programmes. It remains unclear how these mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that single-stranded RNA sequence motifs specifically activate eIF4A1 unwinding allowing local RNA structural rearrangement and translation of eIF4A1-dependent mRNAs in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5'UTR which specifically activate eIF4A1 unwinding of local RNA structure to facilitate translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression, and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex enabling efficient unwinding. Our data proposes a model in which AG-motifs in the 5'UTR of eIF4A1-dependent mRNAs specifically activate eIF4A1, enabling assembly of the helicase-competent multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.