Ultradian rhythms of AKT phosphorylation and gene expression emerge in the absence of the circadian clock components Per1 and Per2

Rhythmicity of biological processes can be elicited either in response to environmental cycles or driven by endogenous oscillators. In mammals, the circadian clock drives about 24-hour rhythms of multitude metabolic and physiological processes in anticipation to environmental daily oscillations. Also at the intersection of environment and metabolism is the protein kinase—AKT. It conveys extracellular signals, primarily feeding-related signals, to regulate various key cellular functions. Previous studies in mice identified rhythmicity in AKT activation (pAKT) with elevated levels in the fed state. However, it is still unknown whether rhythmic AKT activation can be driven through intrinsic mechanisms. Here, we inspected temporal changes in pAKT levels both in cultured cells and animal models. In cultured cells, pAKT levels showed circadian oscillations similar to those observed in livers of wild-type mice under free-running conditions. Unexpectedly, in livers of Per1,2−/− but not of Bmal1−/− mice we detected ultradian (about 16 hours) oscillations of pAKT levels. Importantly, the liver transcriptome of Per1,2−/− mice also showed ultradian rhythms, corresponding to pAKT rhythmicity and consisting of AKT-related genes and regulators. Overall, our findings reveal ultradian rhythms in liver gene expression and AKT phosphorylation that emerge in the absence of environmental rhythms and Per1,2−/− genes.

Cellular model system to dissect the isoform-selectivity of Akt inhibitors

The protein kinase Akt plays a pivotal role in cellular processes. However, its isoforms’ distinct functions have not been resolved to date, mainly due to the lack of suitable biochemical and cellular tools. Against this background, we present the development of an isoform-dependent Ba/F3 model system to translate biochemical results on isoform specificity to the cellular level. Our cellular model system complemented by protein X-ray crystallography and structure-based ligand design results in covalent-allosteric Akt inhibitors with unique selectivity profiles. In a first proof-of-concept, the developed molecules allow studies on isoform-selective effects of Akt inhibition in cancer cells. Thus, this study will pave the way to resolve isoform-selective roles in health and disease and foster the development of next-generation therapeutics with superior on-target properties.

Structure of autoinhibited Akt1 reveals mechanism of PIP3-mediated activation

The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.

Cellular Effects of Rhynchophylline and Relevance to Sleep Regulation

Uncaria rhynchophylla is a plant highly used in the traditional Chinese and Japanese medicines. It has numerous health benefits, which are often attributed to its alkaloid components. Recent studies in humans show that drugs containing Uncaria ameliorate sleep quality and increase sleep time, both in physiological and pathological conditions. Rhynchophylline (Rhy) is one of the principal alkaloids in Uncaria species. Although treatment with Rhy alone has not been tested in humans, observations in rodents show that Rhy increases sleep time. However, the mechanisms by which Rhy could modulate sleep have not been comprehensively described. In this review, we are highlighting cellular pathways that are shown to be targeted by Rhy and which are also known for their implications in the regulation of wakefulness and sleep. We conclude that Rhy can impact sleep through mechanisms involving ion channels, N-methyl-d-aspartate (NMDA) receptors, tyrosine kinase receptors, extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase (PI3K)/RAC serine/threonine-protein kinase (AKT), and nuclear factor-kappa B (NF-κB) pathways. In modulating multiple cellular responses, Rhy impacts neuronal communication in a way that could have substantial effects on sleep phenotypes. Thus, understanding the mechanisms of action of Rhy will have implications for sleep pharmacology.

VEGF Mediates Retinal Müller Cell Viability and Neuroprotection through BDNF in Diabetes

To investigate the mechanism of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) in Müller cell (MC) viability and neuroprotection in diabetic retinopathy (DR), we examined the role of VEGF in MC viability and BDNF production, and the effect of BDNF on MC viability under diabetic conditions. Mouse primary MCs and cells of a rat MC line, rMC1, were used in investigating MC viability and BDNF production under diabetic conditions. VEGF-stimulated BDNF production was confirmed in mice. The mechanism of BDNF-mediated MC viability was examined using siRNA knockdown. Under diabetic conditions, recombinant VEGF (rVEGF) stimulated MC viability and BDNF production in a dose-dependent manner. rBDNF also supported MC viability in a dose-dependent manner. Targeting BDNF receptor tropomyosin receptor kinase B (TRK-B) with siRNA knockdown substantially downregulated the activated (phosphorylated) form of serine/threonine-specific protein kinase (AKT) and extracellular signal-regulated kinase (ERK), classical survival and proliferation mediators. Finally, the loss of MC viability in TrkB siRNA transfected cells under diabetic conditions was rescued by rBDNF. Our results provide direct evidence that VEGF is a positive regulator for BDNF production in diabetes for the first time. This information is essential for developing BDNF-mediated neuroprotection in DR and hypoxic retinal diseases, and for improving anti-VEGF treatment for these blood–retina barrier disorders, in which VEGF is a major therapeutic target for vascular abnormalities.

Regulatory Mechanisms of Autophagy-Targeted Antimicrobial Therapeutics Against Mycobacterial Infection

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen causing human tuberculosis, an infectious disease that still remains as a global health problem. Autophagy, a lysosomal degradative process, has emerged as a critical pathway to restrict intracellular Mtb growth through enhancement of phagosomal maturation. Indeed, several autophagy-modulating agents show promise as host-directed therapeutics for Mtb infection. In this Review, we discuss recent progress in our understanding the molecular mechanisms underlying the action of autophagy-modulating agents to overcome the immune escape strategies mediated by Mtb. The factors and pathways that govern such mechanisms include adenosine 5′-monophosphate-activated protein kinase, Akt/mammalian TOR kinase, Wnt signaling, transcription factor EB, cathelicidins, inflammation, endoplasmic reticulum stress, and autophagy-related genes. A further understanding of these mechanisms will facilitate the development of host-directed therapies against tuberculosis as well as infections with other intracellular bacteria targeted by autophagic degradation.

Akt Is S-Palmitoylated: A New Layer of Regulation for Akt

The protein kinase Akt/PKB participates in a great variety of processes, including translation, cell proliferation and survival, as well as malignant transformation and viral infection. In the last few years, novel Akt posttranslational modifications have been found. However, how these modification patterns affect Akt subcellular localization, target specificity and, in general, function is not thoroughly understood. Here, we postulate and experimentally demonstrate by acyl-biotin exchange (ABE) assay and 3H-palmitate metabolic labeling that Akt is S-palmitoylated, a modification related to protein sorting throughout subcellular membranes. Mutating cysteine 344 into serine blocked Akt S-palmitoylation and diminished its phosphorylation at two key sites, T308 and T450. Particularly, we show that palmitoylation-deficient Akt increases its recruitment to cytoplasmic structures that colocalize with lysosomes, a process stimulated during autophagy. Finally, we found that cysteine 344 in Akt1 is important for proper its function, since Akt1-C344S was unable to support adipocyte cell differentiation in vitro. These results add an unexpected new layer to the already complex Akt molecular code, improving our understanding of cell decision-making mechanisms such as cell survival, differentiation and death.

Bee Venom—A Potential Complementary Medicine Candidate for SARS-CoV-2 Infections

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by severe cytokine storm syndrome following inflammation. SARS-CoV-2 directly interacts with angiotensin-converting enzyme 2 (ACE-2) receptors in the human body. Complementary therapies that impact on expression of IgE and IgG antibodies, including administration of bee venom (BV), have efficacy in the management of arthritis, and Parkinson's disease. A recent epidemiological study in China showed that local beekeepers have a level of immunity against SARS-CoV-2 with and without previous exposure to virus. BV anti-inflammatory properties are associated with melittin and phospholipase A2 (PLA2), both of which show activity against enveloped and non-enveloped viruses, including H1N1 and HIV, with activity mediated through antagonist activity against interleukin-6 (IL-6), IL-8, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). Melittin is associated with the underexpression of proinflammatory cytokines, including nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinases (ERK1/2), and protein kinase Akt. BV therapy also involves group III secretory phospholipase A2 in the management of respiratory and neurological diseases. BV activation of the cellular and humoral immune systems should be explored for the application of complementary medicine for the management of SARS-CoV-2 infections. BV “vaccination” is used to immunize against cytomegalovirus and can suppress metastases through the PLA2 and phosphatidylinositol-(3,4)-bisphosphate pathways. That BV shows efficacy for HIV and H1NI offers opportunity as a candidate for complementary therapy for protection against SARS-CoV-2.