HEK293 Research Articles

Trichostatin A suppresses hearing loss by reducing oxidative stress and inflammation in an Alport syndrome model.

Alport syndrome (AS) is a genetic disorder marked by mutations in type IV collagen, leading to kidney glomerular dysfunction. AS also involves the cochlea, causing late-onset progressive hearing loss. Currently, there are no therapeutic drugs to protect hearing from AS. HDAC inhibitors (HDACis) are chemical compounds that block the activity of histone deacetylase and are known to exert diverse biologic effects. We investigated the effect of Trichostatin A (TSA), an HDACi, to assess its potential to inhibit hearing deterioration in AS. Col4a3 knockout (KO) mice were treated with TSA at 3 weeks of age, and hearing levels were measured using auditory brainstem response (ABR). The results demonstrate that TSA significantly protects the hearing of KO mice compared to the untreated group. The TSA-treated group exhibited a reduction in the levels of oxidative stress markers 4-Hydroxynonenal and 3-Nitrotyrosine, along with a decrease in inflammatory cytokines, in both the mouse cochlea and in vitro HEI-OC1 (House Ear Institute-Organ of Corti 1) cell and HEK (Human Embryonic Kidney)293T cells. AS demonstrated a thickening in the stria vascular vessels, a phenomenon that TSA attenuated. Col4α3 deficiency showed down-regulation of Hemeoxygenase-1 (HO-1), a key anti-inflammatory molecule. TSA treatment induced HO-1 signaling, which contributed to the inhibition of oxidative stress and inflammatory cytokines. These findings suggest that TSA represents a promising candidate molecule for mitigating the progression of hearing loss in AS.

Mycochemistry, antioxidant, anticancer activity, and molecular docking of compounds of F12 of ethyl acetate extract of Astraeus asiaticus with BcL2 and Caspase 3

Globally the current research is going for cancer disease management by different strategies as frequency and severity of this disease are increasing day by day. The fruit body of edible and tasty Astraeus asiaticus mushroom was extracted in ethyl acetate solvent (EA) and quantitative analysis revealed that it contained a significant amount of total phenols, flavonoids, and ascorbic acids. The FT-IR study revealed different functional groups in the extracts with different characteristic peak values. The GC/MS profile of AAEA (Astraeus asiaticus ethyl acetate) extract exhibited 61 compounds. The column chromatography of AAEA extract was performed and the F12 fraction demonstrated the greatest radical scavenging activity with an EC50 of 25.65 ± 4.82 µg. mLˉ¹. Mycochemistry (GC and mass spectrum) analysis showed that F12 was a mixture of six important compounds like Hexadecanoic acid, 3,4,5,6 Tetramethyloctane, 9,12-Octadecadienoic acid, 9,12-Octadecadienoic acid, methyl ester, 1-cyclododecyne, cis-9,10-Epoxyoctadecan-1-ol. The chemical properties of all six compounds were screened by SwissADME and pkCSM and AdmetSAR predictors. Out of them Hexadecanoic acid, 9,12-Octadecadienoic acid and 3,4,5,6 Tetramethyloctane, exhibited suitable properties for drug -preparation and they showed anticancer activity and antioxidant activity as per NIST data base and library search. We have tried to focus on anticancer compounds derived from the partial purification (F12) of mushroom extract (AAEA) from this edible mushroom against cancer (cervical, lung, and breast) cell lines. After 24 h of treatment, the percentages of cell growth inhibition of HeLa, MCF-7, and A549 cell lines by highest concentration (1500 µg. mL− 1) of F12 were 92.03 ± 6.21 a, 90.38 ± 4.53a, and 87.51 ± 5.36a % respectively and the IC50 values were 701 ± 11.54, 728.71 ± 10.53, and 806.88 ± 11.52 µg. mL− 1 respectively but the growth of normal cell HEK 293T was inhibited slightly (3.0%). The mechanism of anticancer effect of F12 (AAEAE) on cancer cell lines included induction of apoptosis, LDH leakage, and up regulation of gene expression levels of Caspase 3, Caspase 9, P53, and down regulation of BcL2 of all three cell lines. Molecular docking of the three important compounds (Hexadecanoic acid, 3,4,5,6 Tetramethyloctane and 9,12-Octadecadienoic acid), with apoptotic protein Caspase 3 and antiapoptotic protein BcL2 was done to find out the binding affinity, stability and drug- likeness properties of these chemicals. In conclusion, F12 fraction of AAEA extract of this mushroom containing six bioactive compounds was a promising antioxidant and anticancer agent and the use of this fraction in cancer treatment will be a novel study for future drug development.

SARS-CoV-2 ORF3a accessory protein is a water-permeable channel that induces lysosome swelling

ORF3a, the most abundantly expressed accessory protein of SARS-CoV-2, plays an essential role in virus egress by inactivating lysosomes through their deacidification. However, the mechanism underlying this process remains unclear. While seminal studies suggested ORF3a being a cation-selective channel (i.e., viroporin), recent works disproved this conclusion. To unravel the potential function of ORF3a, here we employed a multidisciplinary approach including patch-clamp electrophysiology, videoimaging, molecular dynamics (MD) simulations, and electron microscopy. Preliminary structural analyses and patch-clamp recordings in HEK293 cells rule out ORF3a functioning as either viroporin or proton (H+) channel. Conversely, videoimaging experiments demonstrate that ORF3a mediates the transmembrane transport of water. MD simulations identify the tetrameric assembly of ORF3a as the functional water transporter, with a putative selectivity filter for water permeation that includes two essential asparagines, N82 and N119. Consistent with this, N82L and N82W mutations abolish ORF3a-mediated water permeation. Finally, ORF3a expression in HEK293 cells leads to lysosomal volume increase, mitochondrial damage, and accumulation of intracellular membranes, all alterations reverted by the N82W mutation. We propose a novel function for ORF3a as a lysosomal water-permeable channel, essential for lysosome deacidification and inactivation, key steps to promote virus egress.

Construction of a TAT-Cas9-EGFP Site-Specific Integration Eukaryotic Cell Line Using Efficient PEG10 Modification

The CRISPR/Cas9 system enables precise and efficient modification of eukaryotic genomes. Among its various applications, homology-directed repair (HDR) mediated knock-in (KI) is crucial for creating human disease models, gene therapy, and agricultural genetic enhancements. Despite its potential, HDR-mediated knock-in efficiency remains relatively low. This study investigated the impact of 5′ end PEG10 modification on site-specific integration of the target gene. The HEK293 cell line is considered a highly attractive expression system for the production of recombinant proteins, with the construction of site-specific integration cell lines at the AAVS1 locus enabling stable protein expression. This study investigated the impact of the 5′ end PEG10 modification on the site-specific integration of the target gene at the AAVS1 locus in the 293T cell line. Utilizing this 5′ end PEG10 modification resulted in a 1.9-fold increase in knock-in efficiency for a 1.8 kb target fragment, improving efficiency from 26% to 49%. An optimized system was utilized to successfully establish a high-expression, site-specific integration 293T cell line for TAT-Cas9-EGFP, providing a reliable resource of seed cells for subsequent protein production.

Holistic Investigation of Graphene Quantum Dot Endocytosis.

Graphene quantum dots (GQDs) have gained popularity in nano-biotechnology due to their multifunctional delivery and imaging capabilities. The outcome of their therapeutic delivery applications relies on understanding cell internalization routes. Current literature presents often conflicting results based on surveying only a few endocytosis inhibitors. Herein, a holistic approach to cell uptake studies by utilizing six different inhibitors while considering their on- and off-target effects on internalization of the GQDs of different charges is provided. Endocytosis paths are explored by tracking intracellular GQD fluorescence in HeLa or HEK-293 cells. Contrary to the previous assumptions of a singular entry route, findings suggest that GQDs enter the cells through several endocytosis paths with some more prevalent than others. Selectivity between the pathways is based on GQD charge and functional groups. Positively charged nitrogen-doped GQDs (NGQDs) predominantly utilize a fast endophilin-mediated endocytosis (FEME) in HeLa cells with a secondary preference for clathrin-mediated endocytosis (CME). In HEK-293 cells NGQDs internalize via clathrin-independent, glycosylphosphatidylinositol-anchored protein-enriched compartments (CLIC/GEEC) and FEME. Conversely, GQDs with a substantial negative surface charge uptake through CME in HeLa cells. The optimization of these mechanisms can enhance GQD applications in biomedicine, ideally streamlining their translation into the clinic.

Pseudo ternary phase diagram based PEGylated nano-dispersion of linezolid to promote wound regeneration: An in vitro and in vivo evaluation

Open wounds are prone to bacterial infiltration mostly resistant strains like methicillin-resistant Staphylococcus aureus, which affects the healing of open wounds. Topical linezolid nano-dispersion using essential oils as nanoemulgel can increase solubility of the drug and bypass side-effects like GI irritation of oral administration. A Pseudo-ternary phase diagram was built to optimize nanoemulsion. Smix (tween20: PEG400 = 3:1), deionized water, and Oilmix (clove oil:lavender oil = 4:1) with drug was vortexed and then ultrasonicated. 1% carbopol gel of optimized nanoemulsion was prepared and further characterized, then exposed to antimicrobial study, cytocompatibility study using HEK293 cell-line, and in-vivo wound healing study using rat excision model. Histological study was performed to confirm growth of stratum corneum and epithelial tissues. The optimized formulation has particle size(244.6 ± 178.66nm), PDI(25%), entrapment efficiency(92.3 ± 3.38%), and in-vitro drug-release(87.58 ± 4.16%) best fitted in Korsmeyer-Peppas kinetics model(R2=0.9969). NEGF6(0.2%w/w) was found with viscosity of 5345 ± 6cP constituting a very excellent antimicrobial effect against MRSA. HEK293 cells were found viable up to 90% at 800µg/ml indicating good cytocompatibility. The wound contraction rate was 99.66 ± 0.57% at day15 on daily application of 1gm developed gel and the stratum corneum was almost fully regenerated. From the results, we can conclude that developed nanoemulgel has potential antimicrobial efficacy and can promote wound healing.

Highly Stable Antitumor Silver-Lipid Nanoparticles Optimized for Targeted Therapy.

Silver nanoparticles (AgNPs) have a broad spectrum of biocidal effects, allowing also their antitumor application. To enhance bioavailability, minimize adverse effects and enable targeted drug delivery AgNPs may be encapsulated in liposomes. In this study we aimed to create highly stable and effective antitumor AgNP lipid formulations (LAgs). Uncapped and citrate-stabilized AgNPs were encapsulated by the lipid film hydration method using several phospholipid mixtures, followed by the essential removal of unencapsulated AgNPs by size exclusion chromatography (SEC). Purified LAgs were characterized by UV-VIS, DLS, XRD, ICP-MS, transmission electron microscopy (TEM) and glycerol-based density gradient centrifugation (DGC). Liposomal stability was assessed by carboxyfluorescein (CF) leakage, while antitumor effects of purified LAgs were tested in MTT, clonogenic and 3D spheroid invasion experiments. The presence of AgNPs inside SEC-purified liposomes was confirmed by TEM, XRD and ICP-MS. Encapsulation efficiency was estimated to be between 18.7 and 25.5%. Purified LAgs had higher density as compared to free AgNPs revealed by DGC, indicating that a considerable fraction of liposomes contained AgNPs. LAgs with PC/PG, PC/PG/SM/Chol, and in particular PC/PG/SM displayed the highest stability assessed by CF leakage, whereas high content of neutral or negatively charged phospholipids was destabilizing. As shown by MTT and colony formation assays, viability and survival of A375 and RPMI-7951 melanoma cells were severely impaired by LAgs at a higher or comparable level as caused by free AgNPs. Used as a non-tumor control, HEK293 cells were less vulnerable to LAgs as compared to free AgNPs. Finally, applying the most stable lipid composition, PC/PG/SM-LAg-c, and in part PC/PG/SM-LAg-u effectively inhibited a tissue-like invasion of melanoma spheroids. Altogether, highly stable purified LAg formulations were created, which effectively block survival, clonogenic potential and invasion of melanoma cells, therefore could be promising NP platforms for targeted tumor therapy.

Thermoresponsive and cytocompatible infernan-based hydrogels exhibiting poroelastic properties

Thermoresponsive polymers sensitive to temperature changes are particularly well adapted for applications in tissue engineering due to their in situ sol-gel transition at physiological temperature, enabling a rapid gel formation after injection. Infernan, a bacterial exopolysaccharide (EPS) of marine origin endowed with glycosaminoglycan-mimetic properties was grafted with a thermoresponsive polymer, poly(N-isopropylacrylamide) (pNIPAM) using carbodiimide chemistry. The resulting infernan-pNIPAM polymers were shown to form thermoresponsive hydrogels at physiological temperature. Their mechanical properties determined in fully hydrated state at 37°C by Atomic Force Microscopy (AFM) through force-indentation and force-relaxation experiments revealed their poroelastic behavior. Based on AFM measurements, a set of mechanical parameters was extracted, including the elastic modulus, the solvent diffusion coefficient and the pore size. Cytocompatibility of hydrogels evaluated in 3D culture using HEK293 cells was impacted by polysaccharide amount within the hydrogel and the cross-linking density of the network. Elucidation of the structure-function relationship of infernan-pNIPAM hydrogels was essential for their further optimal use in cartilage engineering.

Evaluating the Effectiveness of a Novel Pongamia pinnata Derived Herbal Mouth-Dissolving Film for Treating Oral Disorders and Evaluating Its Anticancer Properties.

The present study aimed to optimize a mouth-dissolving film (MDF) made from Pongamia pinnata stem bark extract to increase patient compliance and accelerate oral disease therapy. Several stem bark extracts were prepared, and karanjin was used as an herbal marker for the extracts. The ethanolic extract showed the maximum yield (12.10% ± 0.09%) and cytotoxic activity against human oral cancer (KB 3-1) and embryonic kidney cell lines. The MDF formulation was focused on incorporating a fixed amount of the extract and varying concentrations of HPMC E5 polymer, along with evaluating the performance of plasticizers like PEG 400 and propylene glycol (PG). An optimized formulation was determined based on disintegration time, wetting time, and folding endurance. The formulation consisted of HPMC E5 as a film-forming polymer, PG as a superior plasticizer, ascorbic acid as an antioxidant, and other ingredients contributing to solubility, dispersion, sweetening, and appearance. High-performance thin-layer chromatography-mass spectrometry analysis confirmed higher levels of karanjin in the optimized formulation, ensuring its successful incorporation and stability. Taste masking evaluations indicate a favorable taste profile and a high potential for patient compliance. The stability study displayed no significant changes in the physical characteristics of the film, affirming its stability and quality. In conclusion, the developed herbal-based optimized MDF presents a promising drug delivery system, offering enhanced patient compliance, taste masking, and stability. The MDF holds great potential for effective treatment and management of oral diseases, providing convenience and improved therapeutic outcomes.

Axin-binding domain of glycogen synthase kinase 3β facilitates functional interactions with voltage-gated Na+channel Nav1.6.

Voltage-gated Na+ (Nav) channels are the primary determinants of the action potential in excitable cells. Nav channels rely on a wide and diverse array of intracellular protein-protein interactions (PPIs) to achieve their full function. Glycogen synthase kinase 3β (GSK3β) has been previously identified as a modulator of Nav1.6-encoded currents and neuronal excitability through PPI formation with Nav1.6 and phosphorylation of its C-terminal domain (CTD). Here, we hypothesized that GSK3β functions as a scaffold in a regulatory PPI complex with the Nav1.6 CTD. Mutagenesis screening using the split-luciferase complementation assay indicated that the axin-binding domain (ABD) of GSK3β (262-299) is necessary for complex formation between the Nav1.6 CTD and GSK3β, and that residues within this domain are drivers of GSK3β-mediated regulation of the channel. Overexpression of an ABD-GFP fusion construct in human embryonic kidney 293cells stably expressing Nav1.6 significantly reduced Nav1.6 nanocluster density compared with GFP alone. In addition, overexpression of the ABD-GFP fusion construct ablates GSK3β-mediated potentiation of Nav1.6-encoded currents and alters channel kinetics. Finally, invivo AAV-mediated overexpression of the ABD-GFP construct in the CA1 hippocampal region induced a reduction in maximal action potential firing and an increase in action potential current threshold in a manner resembling previously reported effects of GSK3β silencing in neurons. Taken together, these results not only suggest that GSK3β-mediated regulation of Nav1.6 extends beyond transient phosphorylation but also implicates the ABD as a critical regulatory domain that facilitates GSK3β's functional effects on Nav1.6 and neuronal excitability.

Phosphorylation of distal C-terminal residues promotes TRPV4 channel activation in response to arachidonic acid.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable channel activated by diverse physical and chemical stimuli, including mechanical stress and endogenous lipid arachidonic acid (AA) and its metabolites. Phosphorylation of TRPV4 by protein kinase A (PKA) and protein kinase C (PKC) is a predominant mechanism for channel regulation, especially in the cytoplasmic domains due to their importance in protein assembly, and channelopathies. However, studies corresponding to phosphorylation sites for these kinases remain incomplete. We investigated the role of Ser-823 residue as a potential phosphorylation site in regulating TRPV4 activity and chemical agonist-induced channel activation. Using mass spectrometry, we identified a new phosphorylation site Ser-823 residue and confirmed the previously known phosphorylation site Ser-824 in the C-terminal tail. The low level of phosphorylation at Ser-823 was stimulated by PKC and to a lesser extent by PKA in human coronary artery endothelial cells (HCAECs) and human embryonic kidney 293 (HEK 293) cells. AA-induced TRPV4 activation was enhanced in the phosphomimetic S823E but was blunted in the S823A/S824A mutants, whereas the channel activation by the synthetic agonist GSK1016790A was unaffected. Further, TRPV4 activation by AA but not GSK1016790A was blunted or abolished by PKA inhibitor alone or in combination with PKC inhibitor, respectively. Using computational modeling, we refined a previously proposed structural model for TRPV4 regulation by Ser-823 and Ser-824 phosphorylation. Together, these results provide insight into how stimuli-specific TRPV4 activation is regulated by the phosphorylation of discrete residues (e.g., Ser-823 and Ser-824) in the C-terminal domains of the TRPV4 channel.

Optimization on cell lysis and capture process of human adenovirus type 5 produced in suspension HEK293 cells

Optimization on cell lysis and capture process of human adenovirus type 5 produced in suspension HEK293 cells

The effect of oxidative stress on the adenosine A2A receptor activity and signalling.

The adenosine A2A receptor (A2AR) is a G-protein coupled receptor that has important anti-inflammatory effects in response to some agonists and consequently is considered a therapeutic target. Its activity is affected by local membrane lipid environment and presence of certain phospholipid classes, so studies should be conducted using extraction methods such as styrene maleic acid co-polymers (SMA) that retain the local lipids. Currently, little is known about the effect of oxidative stress, which may arise from inflammation, on the A2AR. Therefore, it was over-expressed in Pichia pastoris, SMA was used to extract the A2AR from cell membranes and its response to ligands was tested in the presence or absence of the radical initiator AAPH or reactive aldehyde acrolein. SMA-extracted A2AR was able to undergo conformational changes, measured by tryptophan fluorescence, in response to its ligands but oxidative treatments had no effect on the structural changes. Similarly, the treatments did not affect temperature-dependent protein unfolding. In contrast, in HEK293 cells expressing the A2AR, oxidative treatments increased cAMP levels in response to the agonist NECA but had no effect on direct activation of adenylate cyclase. Thus, oxidative stress may be a homeostatic mechanism that abrogates inflammation via the A2AR signalling pathway.

Tetrahydrobiopterin as a rheostat of cell resistance to oxidant injury

Tetrahydrobiopterin (BH4) deficiency is caused by genetic abnormalities that impair its biosynthesis and recycling, which trigger neurochemical, metabolic, and redox imbalances. Low BH4 levels are also associated with hypoxia, reperfusion reoxygenation, endothelial dysfunction, and other conditions that are not genetically determined. The exact cause of changes in BH4 in nongenetic disorders is not entirely understood, but a role for oxidant species has been implicated. The oxidation of BH4 generates several products, including 7,8-dihydrobiopterin (BH2), the accumulation of which is predicted in cells with low dihydrofolate reductase activity. The relative efficiency of oxidant species at causing variations in BH4/BH2 levels in cells furnished with several antioxidant enzymes has not yet been systematically analyzed. This study examined the quantitative changes of BH4/BH2 in cells challenged with several oxidants. We showed that BH2 is not a major product of treatments with hydrogen peroxide or RSL3, as indicated by the moderate effect of dihydrofolate reductase-inhibitor methotrexate on the accumulation of BH2. However, we found a net loss in BH4/BH2, suggesting that products other than BH2 were generated. These reactions were further examined in NOX4-expressing HEK cells producing hydrogen peroxide. These cells showed slightly decreased BH4/BH2 ratios compared with HEK wild-type cells, and, again, methotrexate treatment moderately increased BH2 levels. In contrast, peroxynitrite-producing RAW 264.7 cells showed dramatically decreased BH4 levels without BH2 accumulation. Following the activation of peroxynitrite production with PMA in lipopolysaccharide-treated cells, we also found a significant time-dependent decline in GTPCH-I protein levels. We conclude that hydrogen peroxide is the least effective oxidant species at decreasing intracellular BH4 levels, while peroxynitrite is highly effective by targeting GTPCH-I and BH4 directly. Moreover, we conclude that BH4/BH2 levels are not a determinant of RSL3 cytotoxicity.

MTOR Ser1261 is an AMPK-dependent phosphosite in mouse and human skeletal muscle not required for mTORC2 activity.

The kinases AMPK, and mTOR as part of either mTORC1 or mTORC2, are major orchestrators of cellular growth and metabolism. Phosphorylation of mTOR Ser1261 is reportedly stimulated by both insulin and AMPK activation and a regulator of both mTORC1 and mTORC2 activity. Intrigued by the possibilities that Ser1261 might be a convergence point between insulin and AMPK signaling in skeletal muscle, we investigated the regulation and function of this site using a combination of human exercise, transgenic mouse, and cell culture models. Ser1261 phosphorylation on mTOR did not respond to insulin in any of our tested models, but instead responded acutely to contractile activity in human and mouse muscle in an AMPK activity-dependent manner. Contraction-stimulated mTOR Ser1261 phosphorylation in mice was decreased by Raptor muscle knockout (mKO) and increased by Raptor muscle overexpression, yet was not affected by Rictor mKO, suggesting most of Ser1261 phosphorylation occurs within mTORC1 in skeletal muscle. In accordance, HEK293 cells mTOR Ser1261Ala mutation strongly impaired phosphorylation of mTORC1 substrates but not mTORC2 substrates. However, neither mTORC1 nor mTORC2-dependent phosphorylations were affected in muscle-specific kinase-dead AMPK mice with no detectable mTOR Ser1261 phosphorylation in skeletal muscle. Thus, mTOR Ser1261 is an exercise but not insulin-responsive AMPK-dependent phosphosite in human and murine skeletal muscle, playing an unclear role in mTORC1 regulation but clearly not required for mTORC2 activity.

α-Adrenoreceptor blocker phentolamine inhibits voltage-gated sodium channels via the local anaesthetic binding site.

Phentolamine is a non-selective α-adrenoreceptor antagonist used to reverse local anaesthesia, for example, during dental procedures when a vasoconstrictor is co-applied. Phentolamine-mediated vasodilation leads to faster clearance of injected drugs. Previous electrophysiological studies hypothesized that phentolamine acts as a modulator of voltage-gated sodium channels, which could conflict with its indication as local anaesthetic reversal agent. We performed manual and high throughput patch-clamp recordings on HEK and CHO cells expressing NaV1.7 and NaV1.5. We investigated the effects of phentolamine on sodium channel biophysics and the additive impact of phentolamine on cells preconditioned with the local anaesthetic mexiletine. We used site-directed mutagenesis, homology modelling and drug docking to identify phentolamine's binding site. We compared the effect on sodium channels with other clinically established α-adrenoreceptor antagonists. Phentolamine inhibits NaV1.7 in HEK and CHO cells with an IC50 value of 72 and 57 μM and NaV1.5 in CHO cells with an IC50 of 27 μM. Phentolamine enhances the tonic block induced by the local anaesthetic mexiletine. Phentolamine binds to sodium channels at the local anaesthetic receptor site. The α-adrenoreceptor antagonists alfuzosin, urapidil and phenoxybenzamine show lower potency on NaV1.5 and NaV1.7 in patch-clamp recordings. Phentolamine blocks voltage-gated sodium channels via the local anaesthetic receptor site. This may conflict with its current indication as an antidote for local anaesthetics. We propose alternative α-adrenoreceptor antagonists as possible candidates for local anaesthetic reversal because these are less potent inhibitors of both cardiac and neuronal voltage-gated sodium channels.

SEC-MX: an approach to systematically study the interplay between protein assembly states and phosphorylation

A protein’s molecular interactions and post-translational modifications (PTMs), such as phosphorylation, can be co-dependent and reciprocally co-regulate each other. Although this interplay is central for many biological processes, a systematic method to simultaneously study assembly states and PTMs from the same sample is critically missing. Here, we introduce SEC-MX (Size Exclusion Chromatography fractions MultipleXed), a global quantitative method combining Size Exclusion Chromatography and PTM-enrichment for simultaneous characterization of PTMs and assembly states. SEC-MX enhances throughput, allows phosphopeptide enrichment, and facilitates quantitative differential comparisons between biological conditions. Conducting SEC-MX on HEK293 and HCT116 cells, we generate a proof-of-concept dataset, mapping thousands of phosphopeptides and their assembly states. Our analysis reveals intricate relationships between phosphorylation events and assembly states and generates testable hypotheses for follow-up studies. Overall, we establish SEC-MX as a valuable tool for exploring protein functions and regulation beyond abundance changes.

Pathogenicity assessment of seven RYR1 variants in patients with confirmed susceptibility to malignant hyperthermia in the Netherlands.

Malignant hyperthermia (MH) susceptibility is associated with variants in RYR1, the gene encoding the skeletal muscle ryanodine receptor-1 (RyR1), in 70-75% of patients. Functional characterisation demonstrating an increased sensitivity to RyR1 agonists is necessary among other criteria for inclusion in the European Malignant Hyperthermia Group list of MH susceptibility diagnostic variants. Seven variants in the RYR1 gene, p.Glu342Lys, p.Leu2288Ser, p.Phe2340Leu, p.Arg2676Trp, p.Val3324Ala, p.Phe4076Leu, and p.Trp5020Cys, identified in MH-susceptible individuals were introduced into the cDNA for the human RYR1 gene. These variants were tested in cultured human embryonic kidney HEK293 cells for their effect on calcium release in response to the RyR1 agonist 4-chloro-m-cresol. Calcium release of each variant was compared with wild-type and benign and pathogenic controls. Each variant was subjected to curation using the European Malignant Hyperthermia Group scoring matrix and ClinGen RYR1 Variant Curation Expert Panel guidelines. Six of seven RYR1 variants (p.Glu342Lys, p.Leu2288Ser, p.Phe2340Leu, p.Arg2676Trp, p.Val3324Ala, p.Phe4076Leu) showed hypersensitivity to 4-chloro-m-cresol compared with wild-type. The p.Trp5020Cys variant did not release calcium in response to 4-chloro-m-cresol. All variants had minor allele frequencies <0.1%. Rare exome variant ensemble learner scores of p.Glu342Lys, p.Leu2288Ser, p.Phe4076Leu, and p.Trp5020Cys were >0.85, supporting pathogenicity. The variants p.Glu342Lys, p.Leu2288Ser p.Phe2340Leu, and p.Arg2676Trp are pathogenic or likely pathogenic for MH and can be used for presymptomatic testing for MH susceptibility. As current knowledge on the p.Val3324Ala, p.Phe4076Leu, and p.Trp5020Cys variants remains insufficient, they are still classified as variants of uncertain significance.

A Y178C rhodopsin mutation causes aggregation and comparatively severe retinal degeneration

Rhodopsin is the light-activated G protein-coupled receptor that initiates vision in photoreceptor cells of the retina. Numerous mutations in rhodopsin promote receptor misfolding and aggregation, causing autosomal dominant retinitis pigmentosa, a progressive retinal degenerative disease. The mechanism by which these mutations cause photoreceptor cell death, and the role aggregation plays in this process is still unclear. We recently demonstrated with the P23H and G188R rhodopsin mutants that the severity of aggregation observed in vitro is also reflected in vivo and impacts the rate of retinal degeneration. A Y178C rhodopsin mutant was investigated here to determine if this relationship applies broadly among mutations that cause misfolding and aggregation of the receptor. In vitro characterization indicated the Y178C rhodopsin mutant exhibits similar properties to the more severely aggregating G188R rhodopsin mutant, where the mutant is mislocalized to the endoplasmic reticulum in HEK293 cells and form aggregates that cannot be rescued by treatment with the retinoid 9-cis retinal. Despite these similarities in vitro, the Y178C rhodopsin mutant promoted a more severe retinal degeneration compared to the G188R mutant in vivo in mice. Aggregates of the Y178C rhodopsin mutant labeled by the dye PROTEOSTAT were morphologically similar to those formed by both the P23H and G188R rhodopsin mutants. There was, however, significantly greater photoreceptor cell death occurring independently of PROTEOSTAT-labeled aggregates in mice expressing the Y178C rhodopsin mutant compared to those expressing either the P23H or G188R rhodopsin mutants. Here, we demonstrate that PROTEOSTAT-labeled aggregates are not the sole cause of photoreceptor cell death promoted by the Y178C rhodopsin mutation in vivo, and there may be alternate aggregate forms contributing to cell death in these mice.

Role of Kctd13 in modulating AR and SOX9 expression in different penile cell populations.

Micropenis is a condition with significant physical and psychological implications caused mainly by decreased androgen action in penile development. Kctd13-knockout (Kctd13-KO) mice have micropenis, cryptorchidism, and fertility defects because of reduced levels of androgen receptor (AR) and SOX9. We hypothesized that normalizing the levels of AR and SOX9 in the Kctd13-KO penis could help us to understand the mechanism of action of these signaling pathways on penile development. We generated transgenic mice lacking Kctd13 and conditionally expressing AR in the urethral mesenchyme after Cre activation with Twist2cre (Kctd13-KO; AR-CMV; Twist2cre; herein called AR+), and Sox9 in the urethral epithelium after Cre activation with Shhcre (Kctd13-KO; Sox9-CAG; Shhcre; herein called SOX9+). Mice penile morphology, fertility, and the effect of KCTD13 on AR and SOX9 ubiquitination were evaluated. Kctd13-KO micropenis phenotype was rescued after increasing levels of penile AR or SOX9 as transgenic AR+ and SOX9+ mice have longer penile lengths than Kctd13-KO mice and are comparable to WT mice. In addition, male-urogenital-mating-protuberance and the baculum were significantly shorter and narrower in Kctd13-KO mice compared with transgenic AR+ and SOX9+ mice. The position of the urethral meatus was similar and orthotopic in location in Kctd13-KO, AR+, SOX9+, and WT penises indicating that none of these mice had hypospadias. The subfertility of AR+ and SOX9+ mice was improved. The ectopic expression of KCTD13 in HEK293 cells strongly reduced AR ubiquitination which is abolished when the proteasome pathway is inhibited and this process is mediated by the ubiquitin ligase, STUB1. The effect of KCTD13 on SOX9 ubiquitination is minimal. KCTD13 regulates AR ubiquitination by modulating STUB1 binding to AR. Penile restoration of AR and SOX9 improved penile development in Kctd13-KO mice allowing us to discern the contribution from individual signaling pathways and cell types in penile development.