Intracellular Pool Research Articles

Characterization of a Novel Antimicrobial Peptide Isolated from Moringa oleifera Seed Protein Hydrolysates and Its Membrane Damaging Effects on Staphylococcus aureus.

The present study sought to identify and characterize a novel antimicrobial peptide, named MOp2 from Moringa oleifera seed protein hydrolysates, and elucidate its potential antimicrobial effects on Staphylococcus aureus. MOp2, with the amino acid sequence of His-Val-Leu-Asp-Thr-Pro-Leu-Leu (HVLDTPLL), was characterized as a hydrophobic anionic AMP of the β-sheet structure. MOp2 exhibited negligible hemolytic activity at 2.0× MIC, suggesting its inhibitory effect on the growth of S. aureus (MIC: 2.204 mM). It maintained more than 90% of antimicrobial activity under 5% salt and about 78% of antimicrobial activity at a high temperature of 115 °C for 30 min. Protease, especially acid protease, reduced its antimicrobial activity to different extents. Moreover, MOp2 caused irreversible membrane damage to S. aureus cells by increasing the membrane permeability, resulting in the release of intracellular nucleotide pools. Additionally, molecular docking revealed that MOp2 could inhibit S. aureus growth by interacting with dihydrofolate reductase and DNA gyrase through hydrogen bonding and hydrophobic interactions. Overall, MOp2 could be a potential novel antimicrobial agent against S. aureus in food processing.

A novel viral regulatory network for autophagy induction: Respiratory Syncytial Virus NS2 protein regulates autophagy by modulating BECN1 ISGylation and protein stability.

Respiratory syncytial virus, or RSV, is a leading cause of viral pneumonia and bronchiolitis in children and other susceptible populations. RSV infection dysregulates the immune response leading to exaggerated inflammation in the airway. Among other responses, RSV induces macroautophagy/autophagy, a key process that regulates immune response during infection. We investigated the molecular mechanisms underlying RSV-induced autophagy and showed that the RSV nonstructural NS2 protein promotes autophagy using a dual mechanism. First, NS2 interacts with and stabilizes the autophagy regulator BECN1 (beclin 1), augmenting its intracellular availability for autophagy induction. Second, NS2 interferes with BECN1 ISGylation, thus restricting the intracellular pool of the anti-autophagy ISGylated form of BECN1. Thus, the viral protein (i.e., NS2)-autophagy-ISGylation axis represents a yet unknown regulatory network for viruses. As many viruses induce autophagy that shapes virus-associated immune responses including inflammation, exploring viral protein-autophagy-ISGylation regulatory networks can aid in developing interventions to curb exaggerated immune responses such as inflammation for treating virus-associated inflammatory diseases.

Inflammation and Wasting of Skeletal Muscles in Kras-p53-Mutant Mice with Intraepithelial Neoplasia and Pancreatic Cancer-When Does Cachexia Start?

Skeletal muscle wasting critically impairs the survival and quality of life in patients with pancreatic ductal adenocarcinoma (PDAC). To identify the local factors initiating muscle wasting, we studied inflammation, fiber cross-sectional area (CSA), composition, amino acid metabolism and capillarization, as well as the integrity of neuromuscular junctions (NMJ, pre-/postsynaptic co-staining) and mitochondria (electron microscopy) in the hindlimb muscle of LSL-KrasG12D/+; LSL-TrP53R172H/+; Pdx1-Cre mice with intraepithelial-neoplasia (PanIN) 1-3 and PDAC, compared to wild-type mice (WT). Significant decreases in fiber CSA occurred with PDAC but not with PanIN 1-3, compared to WT: These were found in the gastrocnemius (type 2x: −20.0%) and soleus (type 2a: −21.0%, type 1: −14.2%) muscle with accentuation in the male soleus (type 2a: −24.8%, type 1: −17.4%) and female gastrocnemius muscle (−29.6%). Significantly higher densities of endomysial CD68+ and cyclooxygenase-2+ (COX2+) cells were detected in mice with PDAC, compared to WT mice. Surprisingly, CD68+ and COX2+ cell densities were also higher in mice with PanIN 1-3 in both muscles. Significant positive correlations existed between muscular and hepatic CD68+ or COX2+ cell densities. Moreover, in the gastrocnemius muscle, suppressor-of-cytokine-3 (SOCS3) expressions was upregulated >2.7-fold with PanIN 1A-3 and PDAC. The intracellular pools of proteinogenic amino acids and glutathione significantly increased with PanIN 1A-3 compared to WT. Capillarization, NMJ, and mitochondrial ultrastructure remained unchanged with PanIN or PDAC. In conclusion, the onset of fiber atrophy coincides with the manifestation of PDAC and high-grade local (and hepatic) inflammatory infiltration without compromised microcirculation, innervation or mitochondria. Surprisingly, muscular and hepatic inflammation, SOCS3 upregulation and (proteolytic) increases in free amino acids and glutathione were already detectable in mice with precancerous PanINs. Studies of initial local triggers and defense mechanisms regarding cachexia are warranted for targeted anti-inflammatory prevention.

Structural Investigation of Allosteric Regulation in Class III Ribonucleotide Reductases

Ribonucleotide reductases (RNRs) are essential enzymes that use radical‐based chemistry to catalyze the reduction of ribonucleotides to deoxyribonucleotides. Each RNR class uses a different cofactor to generate a catalytically‐essential thiyl radical species in the enzyme active site. Anaerobic (class III) RNRs employ an oxygen‐sensitive glycyl radical cofactor installed within the glycyl radical domain (GRD) that is adjacent to the active site, whereas aerobic (class Ia) RNRs employ long‐range radical transfer between the catalytic subunit and a di‐iron/tyrosyl radical cofactor housed in the b2 subunit. To maintain intracellular nucleotide pool balance, RNRs are allosterically regulated. For the prototypical class Ia RNRs from Escherichia coli, this allosteric activity regulation involves dATP binding to the N‐terminus of the catalytic subunit in a region known as the ‘cone domain’, resulting in an association between the cone domain and the b2 subunit that prevents radical transfer between subunits. Despite not requiring a b2 subunit, some class III RNRs (e.g. Streptococcus thermophilus)have a cone domain and are allosterically inhibited by dATP. In this study, we investigate allosteric activity regulation for such a class III RNR, the class III RNR from Streptococcus thermophilus(StNrdD). We use a newly developed LC‐MS/MS based activity assay to show that dATP downregulates StNrdD activity and ATP upregulates activity as is true for class Ia RNRs. Using crystallography, we obtain structural data showing that ATP and dATP both bind to the cone domain, and that both the cone domain and GRD are flexible. In particular, in one monomer of StNrdD dimer, the cone domain contacts the GRD, and both domains are ordered, and in the other monomer, the cone domain is positioned away from the GRD and the GRD is disordered. These observations led us to hypothesize that the cone domains of class III RNRs, like those of class Ia RNR, regulate activity through promotion or inhibition of the radical transfer step, but instead of controlling the position of b2, the position of the GRD is allosterically regulated. In this presentation, we show the data described above and show recent cryogenic electron microscopy of ATP‐bound and dATP‐bound states of StNrdD, which reveals differential positioning of the cone domains in the presence of these allosteric effectors. We also present our current thinking about the mechanism of allosteric regulation of activity in a class III RNR.

The Intracellular Cholesterol Pool in Steroidogenic Cells Plays a Role in Autophagy, Basal Steroidogenesis and Mitochondrial Dynamics

In steroidogenesis, cholesterol‐ the precursor substrate molecule for all steroid hormones is central and the framework of steroidogenesis across steroidogenic cells is constructed around it— including its cellular uptake, storage in intracellular lipid droplets, mobilization upon steroidogenic stimulation, and finally, its transport to the mitochondria where cholesterol is converted to pregnenolone by the P450 side chain cleavage enzyme. Thus, cholesterol and the mitochondria are highly interconnected in all steroidogenic cells. Moreover, accruing evidence suggests that autophagy and mitochondrial dynamics are important cellular events in the regulation of trophic hormone‐induced cholesterol homeostasis and steroidogenesis. However, a potential role of cholesterol in the regulation of steroidogenic events and factors remain unexplored. We tested the hypothesis that cholesterol plays a role in the regulation of cell‐intrinsic factors and events involving steroidogenesis. We used an intermediary approach to investigate the role of cholesterol in the regulation of steroidogenesis in different steroidogenic cell types such as MA‐10 cells (a murine Leydig cell line), BeWo cells (placental cell line) producing different steroid hormones. Steroidogenic cells were cultured in a lipoprotein‐depleted medium that leads to the depletion of the intracellular cholesterol pool and lipid droplets. Furthermore, cholesterol deprivation induced steroidogenic events. To get a snapshot of cholesterol deprived (CD)‐induced changes in lipid droplets, autophagy, and mitochondrial dynamics at an ultrastructural level we analyzed steroidogenic cells using transmission electron microscopy. As expected, cholesterol deprivation resulted in the depletion or loss of intracellular lipid droplets and the cholesterol pool across steroidogenic cell lines. Moreover, a difference in autophagy‐related structural changes was observed between cells cultured under the normal and CD conditions. In general, the structural characteristics of autophagy were more apparent in the CD group when compared with the normal control group across different steroidogenic cell types. Moreover, a change in mitochondrial shape was observed between the cells from two groups. For instance, the elongated or tubular mitochondria showing signs of mitochondrial dynamics (fusion or fission) were more common in the CD experimental group. Importantly, an increase in lysosome size and numbers were consistently found in cells under CD conditions which inversely correlated with the depletion of lipid droplets and the cholesterol pool, suggesting a compensatory increase in cholesterol mobilization. Taken together, this data suggests that cholesterol indeed plays a role in the regulation of autophagy and mitochondrial dynamics across steroidogenic cells in a context‐dependent manner. Such effects of cholesterol deprivation on autophagy and mitochondrial dynamics were not observed in the non‐steroidogenic cells such as HepG2 (human hepatoma cells) and H4IIE (rat hepatoma cells), indicating that cholesterol insufficiency‐induced changes in steroidogenic cells are specific to steroidogenesis. Thus, we unraveled this previously unknown role of cholesterol in steroidogenesis beyond being a mere substrate for steroid hormones. The implications of our findings are broad and offer new insights into trophic hormone‐dependent and independent steroidogenesis during development, as well as in health and disease.

Increased Fatty Acid Synthesis and Catabolism Supports Metastatic Breast Cancer Cell Migration

Lipid accumulation is positively associated with breast cancer aggressiveness and poorer clinical outcomes. Despite this observation, mechanisms underlying the increase and role of stored lipid in breast cancer progression remain incompletely understood. The accumulation of triacylglycerol (TAG) is dependent on the balance of uptake, synthesis, and utilization of fatty acids (FAs). In the current study, we utilized non‐metastatic MCF10A‐ras and metastatic MCF10CA1a human breast cancer cells to determine differences in TAG storage and catabolism for sustaining migration—a critical step in the metastatic cascade. Our results demonstrate that MCF10CA1a cells have 90% more TAG than MCF10A‐ras (TAG Assay and Transmission Electron Microscopy), although no significant difference in 14C‐palmitate uptake. MCF10CA1a cells have greater FA synthesis as shown by greater incorporation of 13C‐acetate (60%), 13C‐glucose (65%), and 13C‐glutamine (50%) into palmitate compared to the MCF10A‐ras cells (LC‐MS/MS), as well as higher protein levels of FA synthase (FASN). Additionally, MCF10CA1a cells display greater flux of uniformly‐labeled 13C‐glucose and 13C‐glutamine to the FA synthesis precursor, citrate, and lower intracellular citrate pool size compared to MCF10A‐ras. In addition to increased FA anabolism, MCF10CA1a cells rely on FA oxidation (FAO) for cellular migration compared to MCF10A‐ras cells, as determined by inhibiting the rate‐limiting enzyme of FAO, carnitine palmitoyltransferase 1, with etomoxir (transwell and wound‐healing assays). Pretreatment with the FASN inhibitor TVB‐3166 (3 d) significantly reduced TAG levels and subsequent migration by 67% compared to vehicle‐treated cells. Similarly, pretreatment with inhibitors of the TAG‐synthesizing enzymes diacylglycerol O‐acyltransferase 1 and 2 reduced TAG accumulation and subsequent migration (75%) of MCF10CA1a cells compared to vehicle. Together, the data suggest that FA synthesis may contribute to TAG storage necessary to sustain breast cancer migration. To test this hypothesis, we inhibited the initial enzyme of the TAG lipolysis pathway, adipose triacylglycerol lipase (ATGL; inhibitor ATGListatin), and measured migration. Inhibition of ATGL alone reduced MCF10CA1a cell migration (20% decrease compared to vehicle), and addition of the FAO inhibitor (etomoxir) provided no further effect. Our study indicates that metastatic MCF10CA1a cells accumulate FAs by increasing de novo lipogenesis, store them as TAG, and that catabolism of these stores drives FAO‐dependent migration.

Sources of polyamines in coastal waters and their links to phytoplankton

Polyamines are key components of labile dissolved organic nitrogen in coastal waters. They are found in all living organisms, which are the sources of dissolved organic matter in marine environments. The concentrations of dissolved polyamines are generally low and are controlled as much by production as by consumption. Understanding the dynamics of intracellular pools of polyamines could provide insight to their dynamics in the environment and their potential contribution to ecosystem processes. We measured the concentrations of 6 common polyamines (putrescine, cadaverine, norspermidine, spermidine, norspermine and spermine) extracted from particles in water samples collected on the continental shelf of the South Atlantic Bight (SAB). The total concentrations of extractable polyamines (EPs) decreased from inshore to the shelf-break of the SAB, following a pattern similar to chlorophyll a and opposite to salinity. The composition of EPs was highly correlated with the distribution of phytoplankton species assessed as the abundance of diatom 18S rRNA genes, and with densities of picoeukaryote, Synechococcus and Prochlorococcus cells, suggesting that phytoplankton are the primary source of EPs, followed by a freshwater or nearshore source. Our data suggest that putrescine, norspermidine and spermidine are released primarily from diatoms and picoeukaryotes, while Synechococcus and dinoflagellates are the likely sources of norspermine and spermine.

Catabolic Ornithine Carbamoyltransferase Activity Facilitates Growth of Staphylococcus aureus in Defined Medium Lacking Glucose and Arginine.

ABSTRACTPrevious studies have found that arginine biosynthesis in Staphylococcus aureus is repressed via carbon catabolite repression (CcpA), and proline is used as a precursor. Unexpectedly, however, robust growth of S. aureus is not observed in complete defined medium lacking both glucose and arginine (CDM-R). Mutants able to grow on agar-containing defined medium lacking arginine (CDM-R) were selected and found to contain mutations within ahrC, encoding the canonical arginine biosynthesis pathway repressor (AhrC), or single nucleotide polymorphisms (SNPs) upstream of the native arginine deiminase (ADI) operon arcA1B1D1C1. Reverse transcription-PCR (RT-PCR) studies found that mutations within ccpA or ahrC or SNPs identified upstream of arcA1B1D1C1 increased the transcription of both arcB1 and argGH, encoding ornithine carbamoyltransferase and argininosuccinate synthase/lyase, respectively, facilitating arginine biosynthesis. Furthermore, mutations within the AhrC homologue argR2 facilitated robust growth within CDM-R. Complementation with arcB1 or arcA1B1D1C1, but not argGH, rescued growth in CDM-R. Finally, supplementation of CDM-R with ornithine stimulated growth, as did mutations in genes (proC and rocA) that presumably increased the pyrroline-5-carboxylate and ornithine pools. Collectively, these data suggest that the transcriptional regulation of ornithine carbamoyltransferase and, in addition, the availability of intracellular ornithine pools regulate arginine biosynthesis in S. aureus in the absence of glucose. Surprisingly, ~50% of clinical S. aureus isolates were able to grow in CDM-R. These data suggest that S. aureus is selected to repress arginine biosynthesis in environments with or without glucose; however, mutants may be readily selected that facilitate arginine biosynthesis and growth in specific environments lacking arginine.

HDL, heart disease, and the lung

HDL, heart disease, and the lung

Increased Production of Riboflavin by Coordinated Expression of Multiple Genes in Operons in Bacillus subtilis.

Riboflavin is an essential vitamin widely used in the food, pharmaceutical, and feed industries. However, the insufficient supply of precursors caused by the imbalance of intracellular metabolic flow limits the riboflavin synthesis by industrial strains. Here, we increase riboflavin production by tuning multiple gene expression to balance intracellular metabolic flow. First, we tuned the expression of mCherry and egfp genes within operons by generating libraries of tunable intergenic regions (TIGRs) and confirmed the relative expression of the two reporter genes. The TIGR library can coordinate the expression ratio of reporter genes more than 180 times in Escherichia coli and more than 70 times in Bacillus subtilis. Next, we used this strategy to tune the expression of zwf, ribBA, and ywlf genes within operons through the TIGR library to increase the intracellular precursor pool for riboflavin biosynthesis. Based on the fluorescence characteristics of riboflavin, 96-well plates were used to screen the optimal combination mutants quickly. The best-engineered strain was selected from the library, which produced 2.7 g/L riboflavin, increasing by 64.35% in the shake flask. Finally, the riboflavin titer increased by 59.27% to 11.77 g/L in fed-batch fermentation. The strategy described here will contribute to the industrial production of riboflavin and related products by B. subtilis.

Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation.

Effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By using complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fueled biosynthetic processes while controlling the ratio of succinate/α-ketoglutarate (α-KG) to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells caused proliferation and survival defects when the TCA cycle was truncated, blocking carbon flux to support nucleoside biosynthesis. Replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. SDH deficiency induced a proinflammatory gene signature in T cells and promoted T helper 1 and T helper 17 lineage differentiation. An increasing succinate/α-KG ratio in SDH-deficient T cells promoted inflammation by changing the pattern of the transcriptional and chromatin accessibility signatures and consequentially increasing the expression of the transcription factor, PR domain zinc finger protein 1. Collectively, our studies revealed a role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.

Metabolomics-Driven Identification of the Rate-Limiting Steps in 1-Propanol Production.

The concerted effort for bioproduction of higher alcohols and other commodity chemicals has yielded a consortium of metabolic engineering techniques to identify targets to enhance performance of engineered microbial strains. Here, we demonstrate the use of metabolomics as a tool to systematically identify targets for improved production phenotypes in Escherichia coli. Gas chromatography/mass spectrometry (GC/MS) and ion-pair LC-MS/MS were performed to investigate metabolic perturbations in various 1-propanol producing strains. Two initial strains were compared that differ in the expression of the citramalate and threonine pathways, which hold a synergistic relationship to maximize production yields. While this results in increased productivity, no change in titer was observed when the threonine pathway was overexpressed beyond native levels. Metabolomics revealed accumulation of upstream byproducts, norvaline and 2-aminobutyrate, both of which are derived from 2-ketobutyrate (2KB). Eliminating the competing pathway by gene knockouts or improving flux through overexpression of glycolysis gene effectively increased the intracellular 2KB pool. However, the increase in 2KB intracellular concentration yielded decreased production titers, indicating toxicity caused by 2KB and an insufficient turnover rate of 2KB to 1-propanol. Optimization of alcohol dehydrogenase YqhD activity using an ribosome binding site (RBS) library improved 1-propanol titer (g/L) and yield (g/g of glucose) by 38 and 29% in 72 h compared to the base strain, respectively. This study demonstrates the use of metabolomics as a powerful tool to aid systematic strain improvement for metabolically engineered organisms.

Stochastic parcel tracking in an Euler-Lagrange compartment model for fast simulation of fermentation processes.

The compartment model (CM) is a well‐known approach for computationally affordable, spatially resolved hydrodynamic modeling of unit operations. Recent implementations use flow profiles based on Computational Fluid Dynamics (CFD) simulations, and several authors included microbial kinetics to simulate gradients in bioreactors. However, these studies relied on black‐box kinetics that do not account for intracellular changes and cell population dynamics in response to heterogeneous environments. In this paper, we report the implementation of a Lagrangian reaction model, where the microbial phase is tracked as a set of biomass‐parcels, each linked with an intracellular composition vector and a structured reaction model describing their intracellular response to extracellular variations. A stochastic parcel tracking approach is adopted, in contrast to the resolved trajectories used in CFD implementations. A penicillin production process is used as a case study. We show good performance of the model compared with full CFD simulations, both regarding the extracellular gradients and intracellular pool response, using the mixing time as a matching criterion and taking into account that the mixing time is sensitive to the number of compartments. The sensitivity of the model output towards some of the inputs is explored. The coarsest representative CM requires a few minutes to solve 80 h of flow time, compared with approximately 2 weeks for a full Euler–Lagrange CFD simulation of the same case. This alleviates one of the major bottlenecks for the application of such CFD simulations towards the analysis and optimization of industrial fermentation processes.

Iron Administration Overcomes Resistance to Erastin-Mediated Ferroptosis in Ovarian Cancer Cells.

ObjectivesDeveloping novel therapeutic approaches to defeat chemoresistance is the major goal of ovarian cancer research. Induction of ferroptosis has shown promising antitumor effects in ovarian cancer cells, but the existence of still undefined genetic and metabolic determinants of susceptibility has so far limited the application of ferroptosis inducers in vivo.MethodsErastin and/or the iron compound ferlixit were used to trigger ferroptosis in HEY, COV318, PEO4, and A2780CP ovarian cancer cell lines. Cell viability and cell death were measured by MTT and PI flow cytometry assay, respectively. The “ballooning” phenotype was tested as ferroptosis specific morphological feature. Mitochondrial dysfunction was evaluated based on ultrastructural changes, mitochondrial ROS, and mitochondrial membrane polarization. Lipid peroxidation was tested through both C11-BODIPY and malondialdehyde assays. VDAC2 and GPX4 protein levels were quantified as additional putative indicators of mitochondrial dysfunction or lipid peroxidation, respectively. The effect of erastin/ferlixit treatments on iron metabolism was analyzed by measuring intracellular labile iron pool and ROS. FtH and NCOA4 were measured as biomarkers of ferritinophagy.ResultsHere, we provide evidence that erastin is unable to induce ferroptosis in a series of ovarian cancer cell lines. In HEY cells, provided with a high intracellular labile iron pool, erastin treatment is accompanied by NCOA4-mediated ferritinophagy and mitochondrial dysfunction, thus triggering ferroptosis. In agreement, iron chelation counteracts erastin-induced ferroptosis in these cells. COV318 cells, with low baseline intracellular labile iron pool, appear resistant to erastin treatment. Notably, the use of ferlixit sensitizes COV318 cells to erastin through a NCOA4-independent intracellular iron accumulation and mitochondrial dysfunction. Ferlixit alone mimics erastin effects and promotes ferroptosis in HEY cells.ConclusionThis study proposes both the baseline and the induced intracellular free iron level as a significant determinant of ferroptosis sensitivity and discusses the potential use of ferlixit in combination with erastin to overcome ferroptosis chemoresistance in ovarian cancer.

Screening and Structure-Activity Relationship of D2AAK1 Derivatives for Potential Application in the Treatment of Neurodegenerative Diseases.

Neurodegenerative and mental diseases are serious medical, economic and social problems. Neurodegeneration is referred to as a pathological condition associated with damage to nerve cells leading to their death. Treatment of neurodegenerative diseases is at present symptomatic only, and novel drugs are urgently needed which would be able to stop disease progression. We performed screening of reactive oxygen species, reactive nitrogen species, glutathione and level intracellular Ca2+. The studies were assessed using one-way ANOVA of variance with Dunnett’s post hoc test. Previously, we reported D2AAK1 as a promising compound for the treatment of neurodegenerative and mental disorders. Here, we show a screening of D2AAK1 derivatives aimed at the selection of the compound with the most favorable pharmacological profile. Selected compounds cause an increase in the proliferation of a hippocampal neuron-like cell line, changes in the levels of reactive oxygen and nitrogen forms, reduced glutathione and a reduced intracellular calcium pool. Upon analyzing the structure–activity relationship, we selected the compound with the most favorable profile for a neuroprotective activity for potential application in the treatment of neurodegenerative diseases.

The intracellular cholesterol pool in steroidogenic cells plays a role in basal steroidogenesis

The framework of steroidogenesis across steroidogenic cells is constructed around cholesterol — the precursor substrate molecule for all steroid hormones — including its cellular uptake, storage in intracellular lipid droplets, mobilization upon steroidogenic stimulation, and finally, its transport to the mitochondria, where steroidogenesis begins. Thus, cholesterol and the mitochondria are highly interconnected in steroidogenic cells. Moreover, accruing evidence suggests that autophagy and mitochondrial dynamics are important cellular events in the regulation of trophic hormone-induced cholesterol homeostasis and steroidogenesis. However, a potential role of cholesterol in itself in the regulation of steroidogenic factors and events remain largely unexplored. We tested the hypothesis that cholesterol plays a role in the regulation of cell-intrinsic factors and events involving steroidogenesis. Here, we show that depleting the intracellular cholesterol pool in steroidogenic cells induces autophagy, affects mitochondrial dynamics, and upregulates steroidogenic factors and basal steroidogenesis in three different steroidogenic cell types producing different steroid hormones. Notably, the cholesterol insufficiency-induced changes in different steroidogenic cell types occur independent of pertinent hormone stimulation and work in a dynamic and temporal manner with or without hormonal stimulation. Such effects of cholesterol deprivation on autophagy and mitochondrial dynamics were not observed in the non-steroidogenic cells, indicating that cholesterol insufficiency-induced changes in steroidogenic cells are specific to steroidogenesis. Thus, our data suggests a role of cholesterol in steroidogenesis beyond being a mere substrate for steroid hormones. The implications of our findings are broad and offer new insights into trophic hormone-dependent and hormone-independent steroidogenesis during development, as well as in health and disease.

Alterations in Proteostasis System Components in Peripheral Blood Mononuclear Cells in Parkinson Disease: Focusing on the HSP70 and p62 Levels.

Parkinson disease (PD) is attributed to a proteostasis disorder mediated by α-synuclein accumulating in a specific brain region. PD manifestation is often related to extraneuronal alterations, some of which could be used as diagnostic or prognostic PD biomarkers. In this work, we studied the shifts in the expression of proteostasis-associated chaperones of the HSP70 family and autophagy-dependent p62 protein values in the peripheral blood mononuclear cells (PBMC) of mild to moderate PD patients. Although we did not detect any changes in the intracellular HSP70 protein pool in PD patients compared to non-PD controls, an increase in the transcriptional activity of the stress-associated HSPA1A/B and HSPA6 genes was observed in these cells. Basal p62 content was found to be increased in PD patients’ PBMC, similarly to the p62 level in substantia nigra neural cells in PD. Moreover, the spontaneous apoptosis level was increased among PBMC and positively correlated with the p62 intracellular level in the PD group. A combined HSPA6- and p62-based analysis among 26 PD patients and 36 age-matched non-PD controls pointed out the diagnostic significance of these markers, with intermediate sensitivity and high specificity of this combination when observing patients diagnosed with PD.

Profilin is involved in G1 to S phase progression and mitotic spindle orientation during Leishmania donovani cell division cycle.

Profilin is a multi-ligand binding protein, which is a key regulator of actin dynamics and involved in regulating several cellular functions. It is present in all eukaryotes, including trypanosomatids such as Leishmania. However, not much is known about its functions in these organisms. Our earlier studies have shown that Leishmania parasites express a single homologue of profilin (LdPfn) that binds actin, phosphoinositides and poly- L- proline motives, and depletion of its intracellular pool to 50%of normal levels affects the cell growth and intracellular trafficking. Here, we show, employing affinity pull-down and mass spectroscopy, that LdPfn interacted with a large number of proteins, including those involved in mRNA processing and protein translation initiation, such as eIF4A1. Further, we reveal, using mRNA Seq analysis, that depletion of LdPfn in Leishmania cells (LdPfn+/-) resulted in significantly reduced expression of genes which encode proteins involved in cell cycle regulation, mRNA translation initiation, nucleosides and amino acids transport. In addition, we show that in LdPfn+/- cells, cellular levels of eIF4A1 protein were significantly decreased, and during their cell division cycle, G1-to-S phase progression was delayed and orientation of mitotic spindle altered. These changes were, however, reversed to normal by episomal expression of GFP-LdPfn in LdPfn+/- cells. Taken together, our results indicate that profilin is involved in regulation of G1-to-S phase progression and mitotic spindle orientation in Leishmania cell cycle, perhaps through its interaction with elF4A1 protein.

A Peptide-Based Fluorescent Sensor for Anionic Phospholipids.

Anionic phospholipids are key cell signal mediators. The distribution of these lipids on the cell membrane and intracellular organelle membranes guides the recruitment of signaling proteins leading to the regulation of cellular processes. Hence, fluorescent sensors that can detect anionic phospholipids within living cells can provide a handle into revealing molecular mechanisms underlying lipid-mediated signal regulation. A major challenge in the detection of anionic phospholipids is related to the presence of these phospholipids mostly in the inner leaflet of the plasma membrane and in the membranes of intracellular organelles. Hence, cell-permeable sensors would provide an advantage by enabling the rapid detection and tracking of intracellular pools of anionic phospholipids. We have developed a peptide-based, cell-permeable, water-soluble, and ratiometric fluorescent sensor that entered cells within 15 min of incubation via the endosomal machinery and showed punctate labeling in the cytoplasm. The probe could also be introduced into living cells via lipofection, which allows bypassing of endosomal uptake, to image anionic phospholipids in the cell membrane. We validated the ability of the sensor toward detection of intracellular anionic phospholipids by colocalization studies with a fluorescently tagged lipid and a protein-based anionic phospholipid sensor. Further, the sensor could image the externalization of anionic phospholipids during programmed cell death, indicating the ability of the probe toward detection of both intra- and extracellular anionic phospholipids based on the biological context.

Two alpha isopropylmalate synthase isozymes with similar kinetic properties are extant in the yeast Lachancea kluyveri.

The first committed step in the leucine biosynthetic pathway is catalyzed by α-isopropylmalate synthase (α-IPMS, EC 2.3.3.13), which in the Saccaromycotina subphylum of Ascomycete yeasts is frequently encoded by duplicated genes. Following a gene duplication event, the two copies may be preserved presumably because the encoded proteins diverge in either functional properties and/or cellular localization. The genome of the petite-negative budding yeast Lachancea kluyveri includes two SAKL0E10472 (LkLEU4) and SAKL0F05170g (LkLEU4BIS) paralogous genes, which are homologous to other yeast α-IPMS sequences. Here, we investigate whether these paralogous genes encode functional α-IPMS isozymes and whether their functions have diverged. Molecular phylogeny suggested that the LkLeu4 isozyme is located in the mitochondria and LkLeu4BIS in the cytosol. Comparison of growth rates, leucine intracellular pools and mRNA levels, indicate that the LkLeu4 isozyme is the predominant α-IPMS enzyme during growth on glucose as carbon source. Determination of the kinetic parameters indicates that the isozymes have similar affinities for the substrates and for the feedback inhibitor leucine. Thus, the diversification of the physiological roles of the genes LkLEU4 and LkLEU4BIS involves preferential transcription of the LkLEU4 gene during growth on glucose and different subcellular localization, although ligand interactions have not diverged.