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Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.

Background and aimsIntralipid (ILP) has been demonstrated in animal models and humans to mitigate the cardio-depressant effects of local anesthetics and I/R injury, possibly via restoration of metabolic dysfunction, activation of cardio-protective signaling and augmentation of contractility.Methods8 adult female SD rats received a single intraperitoneal injection of LPS (20 mg/kg). Echocardiography was performed on the rats at baseline prior to injection of LPS, and then at 6h post-LPS, in order to assess LV ejection fraction. Rats were randomly divided to receive either 20% ILP (n=4) or phosphate buffered saline (PBS; n=4) as a 5 ml/kg bolus followed by a 0.5 ml/kg/min infusion over 10 min and echocardiography was conducted at 1, 5 and 10 min to reassess LVEF. At 10 min, LV tissue was collected and Western blots were performed to assess for GSK and STAT3 phosphorylation. Values are expressed as mean±SEM. P<0.05 is considered statistically significant.ResultsBaseline LVEF in PBS and ILP before LPS were 75.7±1.1% and 74.2±1.2%, respectively. 6 h after LPS, LVEF was decreased (LVEF= 54.3±4.8% in PBS, and 46.0±2.5% in ILP; both p<0.05 vs. baseline). Rats treated with ILP had improved EF at 5 min (LVEF=63±3.9% p<0.05 vs.6h post LPS) that peaked at 10 min (LVEF=70.5±2.3%, p<0.05 vs.6h post LPS). PBS group had no significant improvement in LVEF at 5 and 10 min (LVEF=58.4±6.4% and 58.9±7.8%, respectively; both p=ns vs. 6h post LPS). Western blots demonstrated increased phosphorylation of STAT3 (∼2-fold) in ILP treated rats (p<0.05), not GSK phosphorylation.ConclusionsAdministration of ILP significantly improves LVF likely mediated via STAT3 phosphorylation.

T cell exhaustion presents one of the major hurdles to cancer immunotherapy. Among exhausted CD8 + tumor-infiltrating lymphocytes, the terminally exhausted subset contributes directly to tumor cell killing owing to its cytotoxic effector function. However, this subset does not respond to immune checkpoint blockades and is difficult to be reinvigorated with restored proliferative capacity. Here, we show that a half-life-extended interleukin-10–Fc fusion protein directly and potently enhanced expansion and effector function of terminally exhausted CD8 + tumor-infiltrating lymphocytes by promoting oxidative phosphorylation, a process that was independent of the progenitor exhausted T cells. Interleukin-10–Fc was a safe and highly efficient metabolic intervention that synergized with adoptive T cell transfer immunotherapy, leading to eradication of established solid tumors and durable cures in the majority of treated mice. These findings show that metabolic reprogramming by upregulating mitochondrial pyruvate carrier-dependent oxidative phosphorylation can revitalize terminally exhausted T cells and enhance the response to cancer immunotherapy. Tang and colleagues show that a half-life-extended IL-10–Fc fusion protein acts directly on terminally exhausted PD1 + TIM-3 + CD8 + T cells to enhance their proliferation and effector function by reprogramming the cellular metabolism to oxidative phosphorylation in a mitochondrial pyruvate carrier–dependent manner. Treatment of tumor-bearing mice with IL-10–Fc and adoptive T cell therapy led to eradication of their established solid tumors and durable cures.

Background Chronic undernutrition, a condition currently affecting more than 17 millions of children under five years of age, has severe long-term consequences including stunting. Epidemiologic studies have emphasized that undernutrition cannot be ascribed to food insecurity alone and gut microbiota has been shown to play an active role in disease aetiology. In mammals, post-natal growth is controlled by the activity of the somatotropic axis. Undernutrition leads to a decrease of the Insulin like Growth Factor 1 (IGF-1) and a state of Growth Hormon (GH) resistance. Aims Previously we have shown the capacity of selected Lactobacillus plantarum strain to maintain growth in infant mono-colonized mice during chronic undernutrition. Here we show that L. plantarum retains its growth promoting capabilities also in a conventional mouse model. Methods C57BL6 male mice were weaned at 21 days and bred on a standard or an experimental (isocaloric, hypoprotidic and hypolipidic) diet until young adulthood. One group of mice on experimental diet received an LpWJL oral supplementation (2*10^8 CFU/day; 5 days per week) and the other group received placebo. Length and weight were measured weekly. Mice were sacrificed at day 56 to study the impact of the LpWJL oral supplementation on IGF-1 levels and organ growth. Somatotropic axis activity was tested at Day 28 by injecting the mice with GH and measured by the STAT5 phosphorylation level. Results At D56, mice fed with the experimental diet were smaller than the standard diet group (7.7 vs 8.9 cm; p< 0.01). Undernourished mice had a lower hepatic level of IGF-1 (113 ± 39 vs 174 ± 35 pg/mg tissues; p<0.01) and a lower plasmatic level of IGF-1 (150 ± 50 vs 388 ± 103 ng/mL; p< 0.01) compared with the standard diet group. Body length of mice fed experimental diet was longer in LpWJL –supplemented group compared to the placebo supplementation (8.02 ± 0.19 vs 7.73 ± 0.16 cm; p<0.0001). LpWJL-treated mice showed 23% increase in daily growth gain compared to placebo without the change in the mean daily food intake. In the LpWJL group, mice had a higher hepatic IGF-1 level (108 ± 12.5 vs 59.8 ± 18.5 pg/mg tissues; p< 0.0001) and a higher plasmatic IGF-1 level (209 ± 51 vs 148 ± 32 ng/mL; p< 0.001) compared to the placebo group. At day 28, mice exposed to LpWJL during starvation process showed an increase of the sensibility of the hepatic GH receptor to GH according to the STAT5 phosphorylation level. Conclusions Oral supplementation by LpWJL alleviates the GH resistance and improves juvenile growth of conventional infant mice upon undernutrition. Funding Agencies None

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa 1 . However, the irreversible post-mortem degradation 2 of ancient DNA has so far limited its recovery—outside permafrost areas—to specimens that are not older than approximately 0.5 million years (Myr) 3 . By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I 4 , and suggested the presence of protein residues in fossils of the Cretaceous period 5 —although with limited phylogenetic use 6 . In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch 7 – 9 , using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia) 10 . Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros ( Coelodonta antiquitatis ) and Merck’s rhinoceros ( Stephanorhinus kirchbergensis ). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel—which is the hardest tissue in vertebrates 11 , and is highly abundant in the fossil record—can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation. Palaeoproteomic analysis of dental enamel from an Early Pleistocene Stephanorhinus resolves the phylogeny of Eurasian Rhinocerotidae, by enabling the reconstruction of molecular evolution beyond the limits of ancient DNA preservation.

RationaleMaladaptive cardiac hypertrophy predisposes individuals to heart failure. However, current treatments are inaccessible to drugs, and are therefore unsuitable for slowing the progression of hypertrophy. The cell-surface SLC proteins NHE (Slc9a1), NBCe1 (Slc4a4) and NBCn1 (Slc4a7), which are more therapeutically accessible, are critical in neutralising the pH of the highly metabolic cardiomyocytes, and are associated with both physiological and pathophysiological cellular growth. In particular, NBCn1, the electroneutral Na+-HCO3- transporter, is associated with pro-hypertrophic growth in cardiomyocytes and may be of therapeutic interest.MethodsCardiac hypertrophy was evoked in vivo in wildtype or Slc4a7 knockout mice by chemical stressors or abdominal aortic banding (AAB), and changes in cardiac function were assessed by echocardiography. Neonatal rat ventricular myocytes (NRVMs) treated with phenylephrine (PE). Assessment ofResultsNRVMs treated with 10μM PE at pH 7.4 and 24mM HCO3- exhibited increased GATA4 phosphorylation, Anp expression and SRB staining, indicative of increased protein production and consequent hypertrophy. However, these changes were attenuated both at pH 6.4 and in the presence of the pan-NBC inhibitor S0859. However, only Slc4a7 and not Slc4a4 expression was increased in hypertrophic NRVMs, and NBCn1 activity is higher in PE-treated NRVMs at pH 7.4, suggesting that HCO3- transport may be important. In fact, increased PE-induced GATA4 phosphorylation, Anp/Bnp expression and SRB staining was dependent on HCO3-. Moreover, HCO3- was critical for activation of the pro-hypertrophic mTOR signalling cascade and ribosome biogenesis.AAB increased diastolic LV diameter and wall thickness, associated with reduced ejection fraction and fractional shortening; however, these differences were ablated following treatment with S0859. Infusion of isoproterenol via minipumps also resulted in increased myocyte area, and increased Slc4a7 expression and NBCn1 activity in pro-hypertrophic hearts. Additionally, infusion of isoproterenol into Slc4a7 knockout mice exhibited reduced cardiac hypertrophy and left ventricular posterior wall thickness, associated with delayed progression of systolic dysfunction and better cardiac output and stroke volume, demonstrating the importance of the role of NBCn1 in vivo.ConclusionsNBCn1 is the predominant isoform involved in HCO3- dependent alkalinisation and cardiomyocyte hypertrophy, both in vitro and in vivo. It is therefore an attractive therapeutic target for the prevention of cardiac hypertrophy and heart failure.

IntroductionHeart failure (HF) is marked by impaired calcium (Ca2 +) handling, which compromises contractility and heightens arrhythmogenic risk. Ca2 + buffering within cardiac myocytes is essential for stabilizing intracellular Ca2 +. It is underexplored in HF, especially in large-animal models. This study examines how HF alters Ca2 + buffering power and investigates the potential of phosphodiesterase inhibition in restoring Ca2 + dynamics.MethodsHF was induced in adult sheep through tachypacing, and animals were divided into three groups: control, HF (n=10), and HF treated with tadalafil (20 mg daily, n=6). Ventricular cardiac myocytes were isolated, and intracellular Ca2 + was measured using Fluo-5F. Voltage clamp was achieved via a perforated patch clamp. Sarcoplasmic reticulum (SR) Ca2 + content and cellular buffering properties were measured by applying a 5 mM caffeine and 20 mM 2,3-butanedione monoxime solution. Protein levels were quantified using western blotting. All data were analysed with nested statistical models.ResultsHF significantly reduced Ca2 + transient amplitude compared to controls (325.2±34.6nmol/L vs. 169.0±16.8nmol/L, p=0.005), with tadalafil treatment showing no notable improvement (149.4±30.5nmol/L, p=0.96 vs. HF). Ca2 + influx via the L-type Ca2 + channel was also reduced in HF (peak current density 1.26±0.17pA/pF vs. 2.10±0.16pA/pF in controls, p=0.007), this remained impaired in the tadalafil group (1.32±0.22pA/pF, p=0.99 vs. HF). SR Ca2 + content showed no change between controls (77.9±9.6nmol/L) and HF (73.9±9.2nmol/L, p=0.99), but decreased significantly in tadalafil-treated HF (49.0±2.9nmol/L, p=0.018 vs. HF).Ca2 + buffering properties were markedly altered in HF, Bmax decreased from 150.1±19.9µM in controls to 105.2±10.7µM (p=0.02). Kd dropped significantly in HF to 0.61±0.1µM (vs. 1.69±0.20µM in controls, p=0.0002), with an overall increase in buffer power (59.2±6.4 in control and 93.5±7.9 in HF p=0.02). Tadalafil treatment resulted in an increase in Kd to 1.25±0.13 µM (p=0.015 vs. HF), Bmax remained similar to HF at 101.8±4.6 µM (p=0.99 vs. HF). Overall buffer power was restored to baseline levels (61.3±6.5 p=0.02 vs HF.) Western blot analysis showed that HF led to a reduction in TnI phosphorylation (p=0.01), which tadalafil partially restored. SERCA levels were equally reduced in both HF and tadalafil groups (p=0.007 and p=0.003 vs. control, respectively).Abstract BS33 Figure 1Buffer properties with tadalafil treatment. Ai: Ca2+ recordings during application of caffeine + BDM solution. Aii: NCX currents during the same application of caffeine. Aiii: Integral of NCX currents in Aii. Aiv: Representative buffer slopes derived from the caffeine method. Bi: Bmax. Bii: Kd, in control (N=12), HF (N=9) and Tadalafil (N=6) groups. Mean±SEM compared using a nested one-way ANOVA[Image Omitted. See PDF.]Abstract BS33 Figure 2Interplay between buffer power and free and total Ca2+ and tadalafil treatment. Representative trace of free calcium transients (Ai), of total calcium transients (Aii) and the buffer power of cell across the calcium transient (Aiii). Bi: Total calcium amplitude. Bii: Change of total/change of free calcium: ergo cellular buffer power across calcium transient. Biii: Diastolic buffer power. Ci: The relationship between buffer power and free calcium. Cii The relationship between total and free calcium. In control (N=12), HF (N=9) and tadalafil groups (N=6) Mean±SEM compared using nested one-way ANOVA[Image Omitted. See PDF.]ConclusionThis study demonstrates that HF results in an increase in Ca2 + buffering by decreasing Kd, likely due to reduced TnI phosphorylation. Tadalafil restores Ca2 + buffering to baseline by restoring Kd values. This represents a potential therapeutic target for both arrhythmia and contractility in HF. Despite the reduced SR content in tadalafil-treated HF, Ca2 + transient amplitude was preserved, due to the reduction of Ca2 + buffering power seen. These findings propose that targeting Ca2 + buffering with tadalafil may offer a novel strategy to modulate Ca2 + handling in HF.

The transient outward potassium current (Ito) contributes to early repolarisation of the ventricular action potential, controlling action potential duration. Downregulation of Ito, a hallmark of heart failure (HF), prolongs action potential duration (which predisposes the failing heart to lethal cardiac arrhythmias) and adversely affects Ca(v)1.2 activation, subsequently reducing contractility. The channel responsible for Ito is Kv4.3, a voltage sensitive potassium channel, which is palmitoylated at a pair of cysteines in its intracellular C-terminus. We aimed to investigate the cellular control and functional consequences of Kv4.3 palmitoylation.We assessed Kv4.3 interactions (proximity labelling, co-immunoprecipitation), trafficking (membrane impermeable biotinylation reagents), and palmitoylation (acyl-resin assisted capture) in stably transfected cell lines and transiently transfected HEK293 cells.Cardiac Kv4.3 exists as two splice variants whose expression is differentially remodelled during the onset of HF. Kv4.3L contains an additional 19 amino acid in its disordered C-terminus compared to Kv4.3S. We found Kv4.3S is ~2.5–3x more palmitoylated than Kv4.3L and identified key residues within the spliced region that account for the difference in palmitoylation between the variants. The presence of the alternatively spliced region in Kv4.3L reduces its trafficking to the cell surface membrane, which we hypothesize decreases its interaction with the palmitoylating enzyme zDHHC5 and accounts for the reduced palmitoylation. Separately, we identified a phosphorylation site in the Kv4.3 intracellular C-tail near the palmitoylation sites; a non-phosphorylated Kv4.3 mutant is not palmitoylated, suggesting that phosphorylation of this site precedes and is required for Kv4.3 palmitoylation. Preliminary experiments indicate that Kv4.3 phosphorylation promotes its recruitment by zDHHC5, which consequently supports Kv4.3 palmitoylation.In conclusion, the established increase in Kv4.3L and decrease in Kv4.3S expression in HF would result in a net reduction of Kv4.3 palmitoylation in HF. Kv4.3 palmitoylation is dually controlled by alternative splicing and phosphorylation of the Kv4.3 disordered C-tail.

Alzheimer's disease (AD) is the most common cause of dementia, yet there are no drugs available that can prevent or slow disease progression. One reason for this could be because research focussed on treating individuals with mid-to-late AD who present with extensive and potentially irreversible damage. Increasing research is now investigating therapies to target much earlier pathological events in earlier stages of AD. By the point of AD diagnosis, >50% of CA1 hippocampal synapses have already been lost. Furthermore, synaptic plasticity deficits occur before and could be causative of synapse loss. Therefore, understanding the mechanisms driving synaptic plasticity deficits in AD is an attractive area of research that could aid the discovery of new treatments for early AD intervention. One potential candidate contributing to plasticity and synapse loss is Fyn kinase (Fyn), a member of the Src family kinases (SFKs). Post-mortem AD tissue and AD mouse models show increased Fyn activity or phosphorylation of Fyn targets. Fyn phosphorylates Y1472 of GluN2B subunits of the NMDAR, stabilising NMDARs at the synapse. Enhanced Fyn activity in AD is proposed to over-stabilise GluN2B-containing NMDARs and increase NMDAR signalling, which could promote excitotoxicity and contribute to the loss of plasticity and synapses. Using an inducible mouse model of AD (Line 102) to induce APPSwe/Ind expression in adult mice, we analysed mice when synaptic plasticity deficits first arise in CA3-CA1 synapses (after 3 weeks of APPSwe/Ind expression). We show enhanced GluN2B pY1472, consistent with increased Fyn activity. To test whether increased Fyn activity contributes to reduced synaptic plasticity, we measured long-term potentiation (LTP) in hippocampal slices from these mice following inhibition of Fyn with Saracatinib, a drug approved for human use with repurposing potentials for AD. Acute Saracatinib treatment reduced Y1472-GluN2B phosphorylation, and whilst it did not alter LTP in adult WT mice, it rescued the LTP impairment in Line 102 mice. This suggests that increased Fyn activity may contribute to plasticity impairments in this model. However, since Saracatinib is a broad SFK inhibitor, further research is required using more Fyn-specific drugs to determine whether Fyn inhibition is responsible for rescuing plasticity. Acute Saracatinib treatment did not rescue impaired synaptic transmission that occurred later in Line 102 mice, suggesting a narrow therapeutic window of Saracatinib. This thesis supports the evidence suggesting increased Fyn activity in AD, and our results suggest that this increase may occur early in disease progression, possibly before symptom onset. The findings in this project contribute to our understanding of the mechanisms that underlie early AD pathology and add further support to the future use of SFK inhibitors to treat AD.