APX2 Is an Ascorbate Peroxidase-Related Protein that Regulates the Levels of Plastocyanin in Chlamydomonas.

The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from Chlamydomonas reinhardtii, namely, ascorbate peroxidase 2 (APX2). We showed that apx2 mutants exhibited a faster oxidation of the photosystem I primary electron donor, P700, upon sudden light increase and a slower re-reduction rate compared to the wild type, pointing to a limitation of plastocyanin. Spectroscopic, proteomic and immunoblot analyses confirmed that the phenotype was a result of lower levels of plastocyanin in the apx2 mutants. The redox state of P700 did not differ between wild type and apx2 mutants when the loss of function in plastocyanin was nutritionally complemented by growing apx2 mutants under copper deficiency. In this case, cytochrome c6 functionally replaces plastocyanin, confirming that lower levels of plastocyanin were the primary defect caused by the absence of APX2. Overall, the results presented here shed light on an unexpected regulation of plastocyanin level under copper-replete conditions, induced by APX2 in Chlamydomonas.

Transcriptional and translational dynamics underlying heat shock response in the thermophilic crenarchaeon Sulfolobus acidocaldarius.

IMPORTANCE: Heat shock response is the ability to respond adequately to sudden temperature increases that could be harmful for cellular survival and fitness. It is crucial for microorganisms living in volcanic hot springs that are characterized by high temperatures and large temperature fluctuations. In this study, we investigated how S. acidocaldarius, which grows optimally at 75°C, responds to heat shock by altering its gene expression and protein production processes. We shed light on which cellular processes are affected by heat shock and propose a hypothesis on underlying regulatory mechanisms. This work is not only relevant for the organism's lifestyle, but also with regard to its evolutionary status. Indeed, S. acidocaldarius belongs to the archaea, an ancient group of microbes that is more closely related to eukaryotes than to bacteria. Our study thus also contributes to a better understanding of the early evolution of heat shock response.

In-Depth Analysis of the Pancreatic Extracellular Matrix during Development for Next-Generation Tissue Engineering.

The pancreas is a complex organ consisting of differentiated cells and extracellular matrix (ECM) organized adequately to enable its endocrine and exocrine functions. Although much is known about the intrinsic factors that control pancreas development, very few studies have focused on the microenvironment surrounding pancreatic cells. This environment is composed of various cells and ECM components, which play a critical role in maintaining tissue organization and homeostasis. In this study, we applied mass spectrometry to identify and quantify the ECM composition of the developing pancreas at the embryonic (E) day 14.5 and postnatal (P) day 1 stages. Our proteomic analysis identified 160 ECM proteins that displayed a dynamic expression profile with a shift in collagens and proteoglycans. Furthermore, we used atomic force microscopy to measure the biomechanical properties and found that the pancreatic ECM was soft (≤400 Pa) with no significant change during pancreas maturation. Lastly, we optimized a decellularization protocol for P1 pancreatic tissues, incorporating a preliminary crosslinking step, which effectively preserved the 3D organization of the ECM. The resulting ECM scaffold proved suitable for recellularization studies. Our findings provide insights into the composition and biomechanics of the pancreatic embryonic and perinatal ECM, offering a foundation for future studies investigating the dynamic interactions between the ECM and pancreatic cells.

Specific post-translational modifications of soluble tau protein distinguishes Alzheimer's disease and primary tauopathies.

Tau protein aggregates in several neurodegenerative disorders, referred to as tauopathies. The tau isoforms observed in post mortem human brain aggregates is used to classify tauopathies. However, distinguishing tauopathies ante mortem remains challenging, potentially due to differences between insoluble tau in aggregates and soluble tau in body fluids. Here, we demonstrated that tau isoforms differ between tauopathies in insoluble aggregates, but not in soluble brain extracts. We therefore characterized post-translational modifications of both the aggregated and the soluble tau protein obtained from post mortem human brain tissue of patients with Alzheimer's disease, cortico-basal degeneration, Pick's disease, and frontotemporal lobe degeneration. We found specific soluble signatures for each tauopathy and its specific aggregated tau isoforms: including ubiquitination on Lysine 369 for cortico-basal degeneration and acetylation on Lysine 311 for Pick's disease. These findings provide potential targets for future development of fluid-based biomarker assays able to distinguish tauopathies in vivo.

Splenectomy improves erythrocyte functionality in spherocytosis based on septin abundance, but not maturation defects.

Splenectomy improves the clinical parameters of patients with hereditary spherocytosis, but its potential benefit to red blood cell (RBC) functionality and the mechanism behind this benefit remain largely overlooked. Here, we compared 7 nonsplenectomized and 13 splenectomized patients with mutations in the ß-spectrin or the ankyrin gene. We showed that hematological parameters, spherocyte abundance, osmotic fragility, intracellular calcium, and extracellular vesicle release were largely but not completely restored by splenectomy, whereas cryohemolysis was not. Affected RBCs exhibited decreases in ß-spectrin and/or ankyrin contents and slight alterations in spectrin membrane distribution, depending on the mutation. These modifications were found in both splenectomized and nonsplenectomized patients and poorly correlated with RBC functionality alteration, suggesting additional impairments. Accordingly, we found an increased abundance of septins, small guanosine triphosphate-binding cytoskeletal proteins. Septins-2, -7, and -8 but not -11 were less abundant upon splenectomy and correlated with the disease severity. Septin-2 membrane association was confirmed by immunolabeling. Except for cryohemolysis, all parameters of RBC morphology and functionality correlated with septin abundance. The increased septin content might result from RBC maturation defects, as evidenced by (1) the decreased protein 4.2 and Rh-associated glycoprotein content in all patient RBCs, (2) increased endoplasmic reticulum remnants and endocytosis proteins in nonsplenectomized patients, and (3) increased lysosomal and mitochondrial remnants in splenectomized patients. Our study paves the way for a better understanding of the involvement of septins in RBC membrane biophysical properties. In addition, the lack of restoration of septin-independent cryohemolysis by splenectomy may call into question its recommendation in specific cases.

HYGIEIA: HYpothesizing the Genesis of Infectious Diseases and Epidemics through an Integrated Systems Biology Approach.

More than two years on, the COVID-19 pandemic continues to wreak havoc around the world and has battle-tested the pandemic-situation responses of all major global governments. Two key areas of investigation that are still unclear are: the molecular mechanisms that lead to heterogenic patient outcomes, and the causes of Post COVID condition (AKA Long-COVID). In this paper, we introduce the HYGIEIA project, designed to respond to the enormous challenges of the COVID-19 pandemic through a multi-omic approach supported by network medicine. It is hoped that in addition to investigating COVID-19, the logistics deployed within this project will be applicable to other infectious agents, pandemic-type situations, and also other complex, non-infectious diseases. Here, we first look at previous research into COVID-19 in the context of the proteome, metabolome, transcriptome, microbiome, host genome, and viral genome. We then discuss a proposed methodology for a large-scale multi-omic longitudinal study to investigate the aforementioned biological strata through high-throughput sequencing (HTS) and mass-spectrometry (MS) technologies. Lastly, we discuss how a network medicine approach can be used to analyze the data and make meaningful discoveries, with the final aim being the translation of these discoveries into the clinics to improve patient care.

Paladin, overexpressed in colon cancer, is required for actin polymerisation and liver metastasis dissemination.

INTRODUCTION: Colorectal cancer remains a public health issue and most colon cancer patients succumb to the development of metastases. Using a specific protocol of pressure-assisted interstitial fluid extrusion to recover soluble biomarkers, we identified paladin as a potential colon cancer liver metastases biomarker. METHODS: Using shRNA gene knockdown, we explored the biological function of paladin in colon cancer cells and investigated the phospho-proteome within colon cancer cells. We successively applied in vitro migration assays, in vivo metastasis models and co-immunoprecipitation experiments. RESULTS: We discovered that paladin is required for colon cancer cell migration and metastasis, and that paladin depletion altered the phospho-proteome within colon cancer cells. Data are available via ProteomeXchange with identifier PXD030803. Thanks to immunoprecipitation experiments, we demonstrated that paladin, was interacting with SSH1, a phosphatase involved in colon cancer metastasis. Finally, we showed that paladin depletion in cancer cells results in a less dynamic actin cytoskeleton. CONCLUSIONS: Paladin is an undervalued protein in oncology. This study highlights for the first time that, paladin is participating in actin cytoskeleton remodelling and is required for efficient cancer cell migration.

AMPK activation by SC4 inhibits noradrenaline-induced lipolysis and insulin-stimulated lipogenesis in white adipose tissue.

The effects of small-molecule AMP-activated protein kinase (AMPK) activators in rat epididymal adipocytes were compared. SC4 was the most effective and submaximal doses of SC4 and 5-amino-4-imidazolecarboxamide (AICA) riboside were combined to study the effects of AMPK activation in white adipose tissue (WAT). Incubation of rat adipocytes with SC4 + AICA riboside inhibited noradrenaline-induced lipolysis and decreased hormone-sensitive lipase (HSL) Ser563 phosphorylation, without affecting HSL Ser565 phosphorylation. Preincubation of fat pads from wild-type (WT) mice with SC4 + AICA riboside inhibited insulin-stimulated lipogenesis from glucose or acetate and these effects were lost in AMPKα1 knockout (KO) mice, indicating AMPKα1 dependency. Moreover, in fat pads from acetyl-CoA carboxylase (ACC)1/2 S79A/S212A double knockin versus WT mice, the effect of SC4 + AICA riboside to inhibit insulin-stimulated lipogenesis from acetate was lost, pinpointing ACC as the main AMPK target. Treatment with SC4 + AICA riboside decreased insulin-stimulated glucose uptake, an effect that was still observed in fat pads from AMPKα1 KO versus WT mice, suggesting the effect was partly AMPKα1-independent. SC4 + AICA riboside treatment had no effect on the insulin-induced increase in palmitate esterification nor on sn-glycerol-3-phosphate-O-acyltransferase activity. Therefore in WAT, AMPK activation inhibits noradrenaline-induced lipolysis and suppresses insulin-stimulated lipogenesis primarily by inactivating ACC and by inhibiting glucose uptake.

Thiol-disulphide independent in-cell trapping for the identification of peroxiredoxin 2 interactors.

Hydrogen peroxide (H2O2) acts as a signalling molecule by oxidising cysteine thiols in proteins. Recent evidence has established a role for cytosolic peroxiredoxins in transmitting H2O2-based oxidation to a multitude of target proteins. Moreover, it is becoming clear that peroxiredoxins fulfil their function in organised microdomains, where not all interactors are covalently bound. However, most studies aimed at identifying peroxiredoxin interactors were based on methods that only detect covalently linked partners. Here, we explore the applicability of two thiol-disulphide independent in-cell trapping methodological approaches in combination with mass spectrometry for the identification of interaction partners of peroxiredoxin 2 (Prdx2). The first is biotin-dependent proximity-labelling (BioID) with a biotin ligase A (BirA*)-fused Prdx2, which has never been applied on redox-active proteins. The second is crosslinker co-immunoprecipitation with an N-terminally His-tagged Prdx2. During the initial characterisation of the tagged Prdx2 constructs, we found that the His-tag, but not BirA*, compromises the peroxidase and signalling activities of Prdx2. Further, the Prdx2 interactors identified with each approach showed little overlap. We therefore concluded that BioID is a more reliable method than crosslinker co-immunoprecipitation. After a stringent mass spec data filtering, BioID identified 13 interactors under elevated H2O2 conditions, including subunit five of the COP9 signalosome complex (CSN5). The Prdx2:CSN5 interaction was further confirmed in a proximity ligation assay. Taken together, our results demonstrate that BioID can be used as a method for the identification of interactors of Prdxs, and that caution should be exercised when interpreting protein-protein interaction results using tagged Prdxs.

Preclinical Evaluation of the Association of the Cyclin-Dependent Kinase 4/6 Inhibitor, Ribociclib, and Cetuximab in Squamous Cell Carcinoma of the Head and Neck.

Epidermal growth factor receptor (EGFR) overexpression is observed in 90% of human papillomavirus (HPV)-negative squamous cell carcinomas of the head and neck (SCCHN). Cell cycle pathway impairments resulting in cyclin-dependent kinase (CDK) 4 and 6 activation, are frequently observed in SCCHN. We investigated the efficacy of ribociclib, a CDK4/6 inhibitor, in combination with cetuximab, a monoclonal antibody targeting the EGFR, in HPV-negative SCCHN patient-derived tumor xenograft (PDTX) models. The combination of cetuximab and ribociclib was not significantly more active than cetuximab monotherapy in all models investigated. In addition, the combination of cetuximab and ribociclib was less active than ribociclib monotherapy in the cetuximab-resistant PDTX models. In these models, a significant downregulation of the retinoblastoma (Rb) protein was observed in cetuximab-treated mice. We also observed Rb downregulation in the SCCHN cell lines chronically exposed and resistant to cetuximab. In addition, Rb downregulation induced interleukin 6 (Il-6) secretion and the Janus kinase family member/signal transducer and activator of transcription (JAK/STAT) pathway activation that might be implicated in the cetuximab resistance of these cell lines. To conclude, cetuximab is not an appropriate partner for ribociclib in cetuximab-resistant SCCHN models. Our work has significant clinical implications since the combination of anti-EGFR therapy with CDK4/6 inhibitors is currently being investigated in clinical trials.

Aberrant Membrane Composition and Biophysical Properties Impair Erythrocyte Morphology and Functionality in Elliptocytosis.

Red blood cell (RBC) deformability is altered in inherited RBC disorders but the mechanism behind this is poorly understood. Here, we explored the molecular, biophysical, morphological, and functional consequences of α-spectrin mutations in a patient with hereditary elliptocytosis (pEl) almost exclusively expressing the Pro260 variant of SPTA1 and her mother (pElm), heterozygous for this mutation. At the molecular level, the pEI RBC proteome was globally preserved but spectrin density at cell edges was increased. Decreased phosphatidylserine vs. increased lysophosphatidylserine species, and enhanced lipid peroxidation, methemoglobin, and plasma acid sphingomyelinase (aSMase) activity were observed. At the biophysical level, although membrane transversal asymmetry was preserved, curvature at RBC edges and rigidity were increased. Lipid domains were altered for membrane:cytoskeleton anchorage, cholesterol content and response to Ca2+ exchange stimulation. At the morphological and functional levels, pEl RBCs exhibited reduced size and circularity, increased fragility and impaired membrane Ca2+ exchanges. The contribution of increased membrane curvature to the pEl phenotype was shown by mechanistic experiments in healthy RBCs upon lysophosphatidylserine membrane insertion. The role of lipid domain defects was proved by cholesterol depletion and aSMase inhibition in pEl. The data indicate that aberrant membrane content and biophysical properties alter pEl RBC morphology and functionality.

Preclinical Activity of Ribociclib in Squamous Cell Carcinoma of the Head and Neck.

Cell-cycle pathway impairments resulting in CDK4 and 6 activation are frequently observed in human papillomavirus (HPV)-negative squamous cell carcinoma of the head and neck (SCCHN). We investigated the activity of ribociclib, a CDK4/6 inhibitor, in SCCHN models with the aim of identifying predictive biomarkers of response. HPV-negative or HPV-positive SCCHN cell lines (n = 8) and patient-derived tumor xenograft (PDTX) models (n = 6) were used. The models were classified according to their sensitivity to ribociclib to investigate potential predictive biomarkers. Ribociclib had a cytostatic effect in some HPV-negative SCCHN models but had no effect in HPV-positive models. In SCCHN cell lines and PDTXs, the retinoblastoma (Rb) protein expression level correlated with ribociclib activity. Rb knockdown was, however, not sufficient to block G0-G1 arrest induced by ribociclib in Detroit-562 where p107, p130, and Forkhead BOX M1 (FOXM1) were also implicated in ribociclib activity. Cell lines harboring epithelial-to-mesenchymal transition (EMT) features were less sensitive to ribociclib than those with an epithelial phenotype. Rb downregulation induced EMT in our Rb-expressing SCCHN cell lines. However, ribociclib still had significant activity in one PDTX model with high Rb and vimentin expression, suggesting that the presence of vimentin alone is not enough to induce ribociclib resistance. These findings suggest that CDK4/6 inhibitors should be investigated in patients with HPV-negative SCCHN with high Rb expression and an epithelial phenotype. Although these biomarkers are not predictive in all cases, they may enrich the population that could benefit from CDK4/6 inhibitors.

Generation of Organized Porcine Testicular Organoids in Solubilized Hydrogels from Decellularized Extracellular Matrix.

Cryopreservation of immature testicular tissue (ITT) prior to chemo/radiotherapy is now ethically accepted and is currently the only way to preserve fertility of prepubertal boys about to undergo cancer therapies. So far, three-dimensional culture of testicular cells isolated from prepubertal human testicular tissue was neither efficient nor reproducible to obtain mature spermatozoa, and ITT transplantation is not a safe option when there is a risk of cancer cell contamination of the testis. Hence, generation of testicular organoids (TOs) after cell selection is a novel strategy aimed at restoring fertility in these patients. Here, we created TOs using hydrogels developed from decellularized porcine ITT and compared cell numbers, organization and function to TOs generated in collagen only hydrogel. Organotypic culture of porcine ITT was used as a control. Rheological and mass spectrometry analyses of both hydrogels highlighted differences in terms of extracellular matrix stiffness and composition, respectively. Sertoli cells (SCs) and germ cells (GCs) assembled into seminiferous tubule-like structures delimited by a basement membrane while Leydig cells (LCs) and peritubular cells localized outside. TOs were maintained for 45 days in culture and secreted stem cell factor and testosterone demonstrating functionality of SCs and LCs, respectively. In both TOs GC numbers decreased and SC numbers increased. However, LC numbers decreased significantly in the collagen hydrogel TOs (p < 0.05) suggesting a better preservation of growth factors within TOs developed from decellularized ITT and thus a better potential to restore the reproductive capacity.

HOXA2 activity regulation by cytoplasmic relocation, protein stabilization and post-translational modification.

HOX proteins are homeodomain transcription factors critically involved in patterning animal embryos and controlling organogenesis. While the functions of HOX proteins and the processes under their control begin to be well documented, the modalities of HOX protein activity regulation remain poorly understood. Here we show that HOXA2 interacts with PPP1CB, a catalytic subunit of the Ser/Thr PP1 phosphatase complex. This interaction co-localizes in the cytoplasm with a previously described HOXA2 interactor, KPC2, which belongs to the KPC E3 ubiquitin ligase complex. We provide evidence that HOXA2, PPP1CB and KPC2 define a molecularly and functionally interacting complex. Collectively, our experiments support that PPP1CB and KPC2 together inhibit the activity of HOXA2 by activating its nuclear export, but favored HOXA2 de-ubiquitination and stabilization thereby establishing a store of HOXA2 in the cytoplasm.

Eukaryotic elongation factor 2 kinase activity is controlled by multiple inputs from oncogenic signaling.

Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways.

Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase.

eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca(2+) and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr(348), Thr(353), Ser(366) and Ser(445), all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser(78), a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser(78), Thr(348) and Ser(366) to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr(348) was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr(348) appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607-2616]. Ser(366) phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases.

Identification of DH IC-2 as a HIF-1 independent protein involved in the adaptive response to hypoxia in tumor cells: A putative role in metastasis.

The master regulator of the adaptive response to hypoxia is HIF-1. However, while some data show that HIF-1 can control more than 80% of the genes induced under hypoxia, other experiments clearly demonstrate that a part of the hypoxic response is independent of HIF-1. The goal of this study was to identify some of these HIF-1 independent factors and to investigate their functional role in the adaptation of tumor cells to hypoxia. We show that the cytoplasmic dynein intermediate chain 2 (DH IC-2), a component of an intracellular ATPase minus-end directed tubulin-based motile complex, was stabilized and post-translationally modified under hypoxia in a HIF-1 independent way. We identified this modification as a phosphorylation by protein kinase C, which is inhibited under hypoxia. In parallel, the migration of HepG2 cells was enhanced under hypoxia. Cell migration was also increased, to the same extent, by the invalidation of DH IC-2 using siRNA. Taken together, these results suggest that under hypoxia, a specific modification of DH IC-2 may modulate its activity, and in turn promote cell migration. These results are important to better understand cancer development since they highlight a HIF-1 independent mechanism, which may be involved in metastasis.