Nonmuscle myosin IIA dynamically guides regulatory light chain phosphorylation and assembly of nonmuscle myosin IIB

Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II isoforms A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modelling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between isoforms A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-assembles into pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two isoforms form a balanced system for regulated force generation.

Protein Kinase A (PRKA) Activity Is Regulated by the Proteasome at the Onset of Human Sperm Capacitation

The proteasome increases its activity at the onset of sperm capacitation due to the action of the SACY/PRKACA pathway; this increase is required for capacitation to progress. PRKA activity also increases and remains high during capacitation. However, intracellular levels of cAMP decrease in this process. Our goal was to evaluate the role of the proteasome in regulating PRKA activity once capacitation has started. Viable human sperm were incubated in the presence and absence of epoxomicin or with 0.1% DMSO. The activity of PRKA; the phosphorylation pattern of PRKA substrates (pPRKAs); and the expression of PRKAR1, PRKAR2, and AKAP3 were evaluated by Western blot. The localization of pPRKAs, PRKAR1, PRKAR2, and AKAP3 was evaluated by immunofluorescence. Treatment with epoxomicin changed the localization and phosphorylation pattern and decreased the percentage of pPRKAs-positive sperm. PRKA activity significantly increased at 1 min of capacitation and remained high throughout the incubation. However, epoxomicin treatment significantly decreased PRKA activity after 30 min. In addition, PRKAR1 and AKAP3 were degraded by the proteasome but with a different temporal kinetic. Our results suggest that PRKAR1 is the target of PRKA regulation by the proteasome.

Trans-tail regulation-mediated suppression of cryptic transcription

Crosstalk between post-translational modifications of histone proteins influences the regulation of chromatin structure and gene expression. Among such crosstalk pathways, the best-characterized example is H2B monoubiquitination-mediated H3K4 and H3K79 methylation, which is referred to as trans-tail regulation. Although many studies have investigated the fragmentary effects of this pathway on silencing and transcription, its ultimate contribution to transcriptional control has remained unclear. Recent advances in molecular techniques and genomics have, however, revealed that the trans-tail crosstalk is linked to a more diverse cascade of histone modifications and has various functions in cotranscriptional processes. Furthermore, H2B monoubiquitination sequentially facilitates H3K4 dimethylation and histone sumoylation, thereby providing a binding platform for recruiting Set3 complex proteins, including two histone deacetylases, to restrict cryptic transcription from gene bodies. The removal of both ubiquitin and SUMO, small ubiquitin-like modifier, modifications from histones also facilitates a change in the phosphorylation pattern of the RNA polymerase II C-terminal domain that is required for subsequent transcriptional elongation. Therefore, this review describes recent findings regarding trans-tail regulation-driven processes to elaborate on their contribution to maintaining transcriptional fidelity.

Human Recombinant Leptin Shows Dose and Time-Dependent Release of Nitric Oxide from Endothelial Nitric Oxide Synthase in Endothelium While on Angiogenesis

Nitric Oxide (NO) modulates various assortments of the angiogenic process. The endogenous hormone leptin is able to induce different physiological process such as angiogenesis at low concentration because of its high receptor specific affinity. Various studies speculated leptin’s ability to induce endothelium‐dependent vascular relaxation by stimulating NO through different signaling pathways. So far, no studies have reported the dose and time dependent potential of human recombinant leptin on NO release. Hence, an attempt has been made to understand the optimal concentration and time of incubation of human recombinant leptin for the enzymatic release of NO from endothelial Nitric Oxide Synthase (eNOS). Leptin induced changes in the localization and phosphorylation pattern of eNOS in cultured endothelium under various concentrations and time of incubation is studied. The 5 Nanomolar concentration of human recombinant leptin within 6 minutes of incubation could induce significant levels of NO from the activated eNOS in cultured endothelium through plasma membrane localization and phosphorylation of eNOS. Our findings suggest that human recombinant leptin can modulate NO-dependent new therapeutic avenue for angiogenesis-related disorders such as wound healing if used within the active concentration and time of incubation.

Comparative Cytotoxic Activity of Hydroxytyrosol and Its Semisynthetic Lipophilic Derivatives in Prostate Cancer Cells

A high adherence to a Mediterranean diet has been related to numerous beneficial effects in human health, including a lower incidence and mortality of prostate cancer (PCa). Olive oil is an important source of phenolic bioactive compounds, mainly hydroxytyrosol (HT), of this diet. Because of the growing interest of this compound and its derivatives as a cancer chemopreventive agent, we aimed to compare the in vitro effect of HT isolated from olive mill wastewaters and five semisynthetic alkyl ether, ester, and nitro-derivatives against prostate cancer (PCa) cell lines. The effect in cell proliferation was determined in RWPE-1, LNCaP, 22Rv1, and PC-3 cells by resazurin assay, the effect in cell migration by wound healing assay, and tumorsphere and colony formation were evaluated. The changes in key signaling pathways involved in carcinogenesis were assessed by using a phosphorylation pathway profiling array and by Western blotting. Antiproliferative effects of HT and two lipophilic derivatives [hydroxytyrosyl acetate (HT-Ac)/ethyl hydroxytyrosyl ether (HT-Et)] were significantly higher in cancerous PC-3 and 22Rv1 cells than in non-malignant RWPE-1 cells. HT/HT-Ac/HT-Et significantly reduced migration capacity in RWPE-1 and PC-3 and prostatosphere size and colony formation in 22Rv1, whereas only HT-Ac and HT-Et reduced these functional parameters in PC-3. The cytotoxic effect in 22Rv1 cells was correlated with modifications in the phosphorylation pattern of key proteins, including ERK1/2 and AKT. Consistently, HT-Ac and HT-Et decreased p-AKT levels in PC-3. In sum, our results suggest that HT and its lipophilic derivatives could be considered as potential therapeutic tools in PCa.

Functional protection in J20/VLW mice: a model of non-demented with Alzheimer’s disease neuropathology

Alzheimer’s disease comprises amyloid-β and hyperphosphorylated Tau accumulation, imbalanced neuronal activity, aberrant oscillatory rhythms, and cognitive deficits. Non-Demented with Alzheimer’s disease Neuropathology (NDAN) defines a novel clinical entity with amyloid-β and Tau pathologies but preserved cognition. The mechanisms underlying such neuroprotection remain undetermined and animal models of NDAN are currently unavailable. We demonstrate that J20/VLW mice (accumulating amyloid-β and hyperphosphorylated Tau) exhibit preserved hippocampal rhythmic activity and cognition, as opposed to J20 and VLW animals, which show significant alterations. Furthermore, we show that the overexpression of mutant human Tau in coexistence with amyloid-β accumulation renders a particular hyperphosphorylated Tau signature in hippocampal interneurons. The GABAergic septohippocampal pathway, responsible for hippocampal rhythmic activity, is preserved in J20/VLW mice, in contrast to single mutants. Our data highlight J20/VLW mice as a suitable animal model in which to explore the mechanisms driving cognitive preservation in NDAN. Moreover, they suggest that a differential Tau phosphorylation pattern in hippocampal interneurons prevents the loss of GABAergic septohippocampal innervation and alterations in local field potentials, thereby avoiding cognitive deficits.

Localization and phosphorylation in the Snf1 network is controlled by two independent pathways

AMPK/SNF1 is the master regulator of energy homeostasis in eukaryotic cells and has a key role in glucose de-repression. If glucose becomes depleted, Snf1 is phosphorylated and activated. Activation of Snf1 is required but is not sufficient for mediating glucose de-repression indicating a second glucose-regulated step that adjusts the Snf1 pathway. To elucidate this regulation, we further explore the spatial dynamics of Snf1 and Mig1 and how they are controlled by concentrations of hexose sugars. We utilize fluorescence recovery after photobleaching (FRAP) to study the movement of Snf1 and how it responds to external glucose concentrations. We show that the Snf1 pathway reacts both to the presence and to the absolute concentration of glucose. Furthermore, we identify a negative feedback loop regulating Snf1 activity. We also show that Mig1 localization correlates with the Snf1 phosphorylation pattern and not with the Mig1 phosphorylation pattern, suggesting that inactivation of Snf1 has a more pronounced effect on the localization of Mig1 than on the phosphorylation of Mig1. Our data offer insight into the true complexity of regulation of this central signaling pathway by one signal (glucose depletion) interpreted by the cell in different ways.

Copper Dependent Modulation of α-Synuclein Phosphorylation in Differentiated SHSY5Y Neuroblastoma Cells

Copper (Cu) dyshomeostasis plays a pivotal role in several neuropathologies, such as Parkinson’s disease (PD). Metal accumulation in the central nervous system (CNS) could result in loss-of-function of proteins involved in Cu metabolism and redox cycling, generating reactive oxygen species (ROS). Moreover, neurodegenerative disorders imply the presence of an excess of misfolded proteins known to lead to neuronal damage. In PD, Cu accumulates in the brain, binds α-synuclein, and initiates its aggregation. We assessed the correlation between neuronal differentiation, Cu homeostasis regulation, and α-synuclein phosphorylation. At this purpose, we used differentiated SHSY5Y neuroblastoma cells to reproduce some of the characteristics of the dopaminergic neurons. Here, we reported that differentiated cells expressed a significantly higher amount of a copper transporter protein 1 (CTR1), increasing the copper uptake. Cells also showed a significantly more phosphorylated form of α-synuclein, further increased by copper treatment, without modifications in α-synuclein levels. This effect depended on the upregulation of the polo-like kinase 2 (PLK2), whereas the levels of the relative protein phosphatase 2A (PP2A) remained unvaried. No changes in the oxidative state of the cells were identified. The Cu dependent alteration of α-synuclein phosphorylation pattern might potentially offer new opportunities for clinical intervention.

Salt induced programmed cell death in rice: evidence from chloroplast proteome signature

Soil salinity, depending on its intensity, drives a challenged plant either to death, or survival with compromised productivity. On exposure to moderate salinity, plants can often survive by sacrificing some of their cells ‘in target’ following a route called programmed cell death (PCD). In animals, PCD has been well characterised, and involvement of mitochondria in the execution of PCD events has been unequivocally proven. In plants, mechanistic details of the process are still in grey area. Previously, we have shown that in green tissues of rice, for salt induced PCD to occur, the presence of active chloroplasts and light are equally important. In the present work, we have characterised the chloroplast proteome in rice seedlings at 12 and 24 h after salt exposure and before the time point where the signature of PCD was observed. We identified almost 100 proteins from chloroplasts, which were divided in to 11 categories based on the biological functions in which they were involved. Our results concerning the differential expression of chloroplastic proteins revealed involvement of some novel candidates. Moreover, we observed maximum phosphorylation pattern of chloroplastic proteins at an early time point (12 h) of salt exposure.