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CRISPR‐Cas9 screens facilitate the discovery of gene functional relationships and phenotype‐specific dependencies. The Cancer Dependency Map (DepMap) is the largest compendium of whole‐genome CRISPR screens aimed at identifying cancer‐specific genetic dependencies across human cell lines. A mitochondria‐associated bias has been previously reported to mask signals for genes involved in other functions, and thus, methods for normalizing this dominant signal to improve co‐essentiality networks are of interest. In this study, we explore three unsupervised dimensionality reduction methods—autoencoders, robust, and classical principal component analyses (PCA)—for normalizing the DepMap to improve functional networks extracted from these data. We propose a novel "onion" normalization technique to combine several normalized data layers into a single network. Benchmarking analyses reveal that robust PCA combined with onion normalization outperforms existing methods for normalizing the DepMap. Our work demonstrates the value of removing low‐dimensional signals from the DepMap before constructing functional gene networks and provides generalizable dimensionality reduction‐based normalization tools. Synopsis: Dimensionality reduction‐based methods are proposed for normalizing Cancer Dependency Map (DepMap) genome‐wide CRISPR screen data to enhance the functional information in co‐essentiality networks extracted from DepMap.Low‐dimensional patterns introduce dominant covariation in gene networks derived from DepMap data, obscuring more subtle functional relationships.Applying dimensionality reduction approaches to remove low‐dimensional signal, including robust and classical principal component analysis or autoencoders, can increase functional information captured by similarity networks derived from DepMap data.Onion normalization, which integrates several normalized data layers into a single network, outperforms existing methods for constructing co‐essentiality networks from the DepMap. [ABSTRACT FROM AUTHOR]

Transcription factor RUNX1 plays important roles in hematopoiesis and leukemogenesis. RUNX1 function is tightly controlled through posttranslational modifications, including ubiquitination and acetylation. However, its regulation via ubiquitination, especially proteasome‐independent ubiquitination, is poorly understood. We previously identified DTX2 as a RUNX1‐interacting E3 ligase using a cell‐free AlphaScreen assay. In this study, we examined whether DTX2 is involved in the regulation of RUNX1 using in vitro and ex vivo analyses. DTX2 bound to RUNX1 and other RUNX family members RUNX2 and RUNX3 through their C‐terminal region. DTX2‐induced RUNX1 ubiquitination did not result in RUNX1 protein degradation. Instead, we found that the acetylation of RUNX1, which is known to enhance the transcriptional activity of RUNX1, was inhibited in the presence of DTX2. Concomitantly, DTX2 reduced the RUNX1‐induced activation of an MCSFR luciferase reporter. We also found that DTX2 induced RUNX1 cytoplasmic mislocalization. Moreover, DTX2 overexpression showed a substantial growth‐inhibitory effect in RUNX1‐dependent leukemia cell lines. Thus, our findings indicate a novel aspect of the ubiquitination and acetylation of RUNX1 that is modulated by DTX2 in a proteosome‐independent manner. [ABSTRACT FROM AUTHOR]

The article focuses on the characterization of claudin-low breast cancer, which is a subtype mainly characterized by being negative for estrogen (ER), progesterone (PR), and HER2 receptors. Topics include the molecular characteristics of claudin-low breast cancer cell lines, their gene dependencies, and drug sensitivities, suggesting potential treatments worth exploring for this subtype of breast cancer, which is known for its limited therapeutic options.

Quantitative microscopy is a powerful method for performing phenotypic screens from which image‐based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image‐based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments. Here, we examine the performance of the Cell Painting assay across multiple high‐throughput microscope systems and find that all are compatible with this assay. Furthermore, we determine independently for each microscope system the best performing settings, providing those who wish to adopt this assay an ideal starting point for their own assays. We also explore the impact of microscopy setting changes in the Cell Painting assay and find that few dramatically reduce the quality of a Cell Painting profile, regardless of the microscope used. [ABSTRACT FROM AUTHOR]

RAF activation is a key step for signalling through the mitogen‐activated protein kinase (MAPK) pathway. The SHOC2 protein, along with MRAS and PP1C, forms a high affinity, heterotrimeric holoenzyme that activates RAF kinases by dephosphorylating a specific phosphoserine. Recently, our research, along with that of three other teams, has uncovered valuable structural and functional insights into the SHOC2‐MRAS‐PP1C (SMP) holoenzyme complex. In this structural snapshot, we review SMP complex assembly, the dependency on the bound‐nucleotide state of MRAS, the substitution of MRAS by the canonical RAS proteins and the roles of SHOC2 and MRAS on PP1C activity and specificity. Furthermore, we discuss the effect of several RASopathy mutations identified within the SMP complex and explore potential therapeutic approaches for targeting the SMP complex in RAS/RAF‐driven cancers and RASopathies. [ABSTRACT FROM AUTHOR]

Background: Genetic suppression occurs when the deleterious effects of a primary "query" mutation, such as a disease-causing mutation, are rescued by a suppressor mutation elsewhere in the genome. Methods: To capture existing knowledge on suppression relationships between human genes, we examined 2,400 published papers for potential interactions identified through either genetic modification of cultured human cells or through association studies in patients. Results: The resulting network encompassed 476 unique suppression interactions covering a wide spectrum of diseases and biological functions. The interactions frequently linked genes that operate in the same biological process. Suppressors were strongly enriched for genes with a role in stress response or signaling, suggesting that deleterious mutations can often be buffered by modulating signaling cascades or immune responses. Suppressor mutations tended to be deleterious when they occurred in absence of the query mutation, in apparent contrast with their protective role in the presence of the query. We formulated and quantified mechanisms of genetic suppression that could explain 71% of interactions and provided mechanistic insight into disease pathology. Finally, we used these observations to predict suppressor genes in the human genome. Conclusions: The global suppression network allowed us to define principles of genetic suppression that were conserved across diseases, model systems, and species. The emerging frequency of suppression interactions among human genes and range of underlying mechanisms, together with the prevalence of suppression in model organisms, suggest that compensatory mutations may exist for most genetic diseases. [ABSTRACT FROM AUTHOR]

Background: Solasonine, as a major biological component of Solanum nigrum L., has demonstrated anticancer effects against several malignancies. However, little is understood regarding its biological target and mechanism in non-small cell lung cancer (NSCLC). Methods: We conducted an analysis on transcriptomic data to identify differentially expressed genes (DEGs), and employed an artificial intelligence (AI) strategy to predict the target protein for solasonine. Subsequently, genetic dependency analysis and molecular docking were performed, with Acetylcholinesterase (ACHE) selected as a pivotal marker for solasonine. We then employed a range of bioinformatic approaches to explore the relationship between ACHE and solasonine. Furthermore, we investigated the impact of solasonine on A549 cells, a human lung cancer cell line. Cell inhibition of A549 cells following solasonine treatment was analyzed using the CCK8 assay. Additionally, we assessed the protein expression of ACHE, as well as markers associated with apoptosis and inflammation, using western blotting. To investigate their functions, we employed a plasmid-based ACHE overexpression system. Finally, we performed dynamics simulations to simulate the interaction mode between solasonine and ACHE. Results: The results of the genetic dependency analysis revealed that ACHE could be identified as the pivotal target with the highest docking affinity. The cell experiments yielded significant findings, as evidenced by the negative regulatory effect of solasonine treatment on tumor cells, as demonstrated by the CCK8 assay. Western blotting analysis revealed that solasonine treatment resulted in the downregulation of the Bcl-2/Bax ratio and upregulation of cleaved caspase-3 protein expression levels. Moreover, we observed that ACHE overexpression promoted the expression of the Bcl-2/Bax ratio and decreased cleaved caspase-3 expression in the OE-ACHE group. Notably, solasonine treatment rescued the Bcl-2/Bax ratio and cleaved caspase-3 expression in OE-ACHE cells compared to OE-ACHE cells without solasonine treatment, suggesting that solasonine induces apoptosis. Besides, solasonine exhibited its anti-inflammatory effects by inhibiting P38 MAPK. This was supported by the decline in protein levels of IL-1β and TNF-α, as well as the phosphorylated forms of JNK and P38 MAPK. The results from the molecular docking and dynamics simulations further confirmed the potent binding affinity and effective inhibitory action between solasonine and ACHE. Conclusions: The findings of the current investigation show that solasonine exerts its pro-apoptosis and anti-inflammatory effects by suppressing the expression of ACHE. [ABSTRACT FROM AUTHOR]

Background: While the increased screening, changes in lifestyle, and recent advances in treatment regimen have decreased colorectal cancer (CRC) mortality, metastatic disease and recurrence remains a major clinical challenge. In the era of precision medicine, the identification of actionable novel therapeutic targets could ultimately offer an alternative treatment strategy for CRC. Methods: RNA-Seq was conducted using the illumina platform, while bioinformatics analyses were conducted using CLC genomics workbench and iDEP.951. Colony forming unit, flow cytometry, and fluorescent microscopy were used to assess cell proliferation, cell cycle distribution, and cell death, respectively. The growth potential of CRC cells under 3-dimensional (3D) conditions was assessed using Matrigel. STRING database (v11.5) and Ingenuity Pathway Analysis (IPA) tool were used for network and pathway analyses. CRISPR-Cas9 perturbational effects database was used to identify potential therapeutic targets for CRC, through integration with gene-drug interaction database. Structural modeling and molecular docking were used to assess the interaction between candidate drugs and their targets. Results: In the current study, we investigated the therapeutic potential of targeting TPX2, TTK, DDX39A, and LRP8, commonly upregulated genes in CRC identified through differential expression analysis in CRC and adjacent non-cancerous tissue. Targeted depletion of TPX2 and TTK impaired CRC proliferation, cell cycle progression, and organoid formation under 3D culture conditions, while suppression of DDX39A and LRP8 had modest effects on CRC colony formation. Differential expression analysis and bioinformatics on TPX2 and TTK-deficient cells identified cell cycle regulation as the hallmark associated with loss of TPX2 and TTK. Elevated expression of TPX2 and TTK correlated with an oncogenic state in tumor tissue from patients with colon adenocarcinoma, thus corroborating an oncogenic role for the TPX2/TTK network in the pathogenesis of CRC. Gene set enrichment and pathway analysis of TPX2high/TTKhigh CRC identified numerous additional gene targets as integral components of the TPX2/TTK network. Integration of TPX2/TTK enriched network with CRISPR-Cas9 functional screen data identified numerous novel dependencies for CRC. Additionally, gene-drug interaction analysis identified several druggable gene targets enriched in the TPX2/TTK network, including AURKA, TOP2A, CDK1, BIRC5, and many others. Conclusions: Our data has implicated an essential role for TPX2 and TTK in CRC pathogenesis and identified numerous potential therapeutic targets and their drug interactions, suggesting their potential clinical use as a novel therapeutic strategy for patients with CRC. 8pAfbPjv5QF2m5WMY3QXvq Video Abstract [ABSTRACT FROM AUTHOR]

Mutually exclusive loss-of-function alterations in gene pairs are those that occur together less frequently than may be expected and may denote a synthetically lethal relationship (SSL) between the genes. SSLs can be exploited therapeutically to selectively kill cancer cells. Here, we analysed mutation, copy number variation, and methylation levels in samples from The Cancer Genome Atlas, using the hypergeometric and the Poisson binomial tests to identify mutually exclusive inactivated genes. We focused on gene pairs where one is an inactivated tumour suppressor and the other a gene whose protein product can be inhibited by known drugs. This provided an abundance of potential targeted therapeutics and repositioning opportunities for several cancers. These data are available on the MexDrugs website, https://bioinformaticslab.sussex.ac.uk/mexdrugs., (© 2024 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The modification of the triple helical structure in aqueous gelatin solutions by changing pH and adding alkyl sulphates at 298 K and after rechilling the solution to 283 K was investigated by CD-measurement. At 298 K the triple helical content at the IEP of the gelatin has its maximum value. It is only weakly affected by adding sodium dodecyl sulphate (SDDS) at concentrations <10 M/dm. The unfolding of the triple helix affected by pH and SDDS is reversible by rechilling the solution. The triple helical content of gelatin solutions decreases at SDDS concentrations higher than 10 M/dm. In all cases the decrease of the amount of triple helical structure is connected with an increase of the cis-configuration in single chains and leads to chain reversals. At sufficiently high SDDS concentrations β-sheets are formed. These changes are thermally irreversible. Sodium decyl sulphate (SDS) has a more minor influence than SDDS except in the range of the c.m.c. of SDS. At sufficiently high SDS concentrations, β-turns appear. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma (GBM) is a type of highly malignant brain tumor that is known for its significant intratumoral heterogeneity, meaning that there can be a high degree of variability within the tumor tissue. Despite the identification of several subtypes of GBM in recent years, there remains to explore a classification based on genes related to proliferation and growth. Methods: The growth-related genes of GBM were identified by CRISPR-Cas9 and univariate Cox regression analysis. The expression of these genes in the Cancer Genome Atlas cohort (TCGA) was used to construct growth-related genes subtypes (GGSs) via consensus clustering. Validation of this subtyping was performed using the nearest template prediction (NTP) algorithm in two independent Gene Expression Omnibus (GEO) cohorts and the ZZ cohort. Additionally, copy number variations, biological functions, and potential drugs were analyzed for each of the different subtypes separately. Results: Our research established multicenter-validated GGSs. GGS1 exhibits the poorest prognosis, with the highest frequency of chr 7 gain & chr 10 loss, and the lowest frequency of chr 19 & 20 co-gain. Additionally, GGS1 displays the highest expression of EGFR. Furthermore, it is significantly enriched in metabolic, stemness, proliferation, and signaling pathways. Besides we showed that Foretinib may be a potential therapeutic agent for GGS1, the worst prognostic subtype, through data screening and in vitro experiments. GGS2 has a moderate prognosis, with a slightly higher proportion of chr 7 gain & chr 10 loss, and the highest proportion of chr 19 & 20 co-gain. The prognosis of GGS3 is the best, with the least chr 7 gain & 10 loss and EGFR expression. Conclusions: These results enhance our understanding of the heterogeneity of GBM and offer insights for stratified management and precise treatment of GBM patients. [ABSTRACT FROM AUTHOR]

GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein translation in response to stress. Here, we show that GCN2 has an unexpected role in unstressed cells as a regulator of mitosis. This function is not through its canonical role in translation reprogramming, but through the regulation of two previously unidentified substrates, PP1α and γ. In the absence of GCN2 function, timing and levels of phosphorylation of key mitotic players are altered, leading to aberrant chromosome alignment, missegregating chromosomes, elevated number of tripolar spindles, and a delay in progression through mitosis. Pharmacological inhibition of GCN2 results in similar effects and is synergistic with Aurora A inhibition in causing more severe mitotic errors and cell death. We suggest that GCN2‐dependent phosphorylation of PP1α and γ restrains their activity and this is important to ensure the timely regulation of phosphorylation of several PP1 substrates during early mitosis. These findings highlight a druggable PP1 inhibitor and open new avenues of research on the therapeutic potential of GCN2 inhibitors. Synopsis: GCN2 phosphorylates novel substrates to ensure error‐free chromosome alignment in mitosis. Depletion or chemical inhibition of GCN2 in cancer cells results in a delay in mitotic progression.The mitotic function of GCN2 is not through its canonical role in translation reprogramming, but through the regulation of PP1α and γ.GCN2 restrains the activity of PP1α and γ during early mitosis to ensure the timely regulation of phosphorylation of several PP1 substrates.Pharmacological inhibition of GCN2 is synergistic with Aurora A inhibition in causing more severe mitotic errors and cell death. [ABSTRACT FROM AUTHOR]

Cell painting technique provides large amount of potential information for applications such as drug discovery, bioactivity prediction and cytotoxicity assessment. However, its utility is restricted due to the requirement of advanced, costly and specific instrumentation protocols. Therefore, creating cell painted images using simple microscopic data can provide a better alternative for these applications. This study investigates the applicability of deep network-based semantic segmentation to generate cell painted images of nuclei, endoplasmic reticulum (ER) and cytoplasm from a composite image. For this, 3456 composite images from a public dataset of Broad Bioimage Benchmark collection are considered. The corresponding ground truth images for nuclei, ER and cytoplasm are generated using Otsu's thresholding technique and used as labeled dataset. Semantic segmentation network is applied to these data and optimized using stochastic gradient descent with momentum algorithm at a learning rate of 0.01. The segmentation performance of the trained network is evaluated using accuracy, loss, mean Boundary F (BF) score, Dice Index, Jaccard Index and structural similarity index. Gradient weighted Class Activation Mapping (Grad-CAM) is employed to visualize significant image regions identified by the model. Further, a cellular index is proposed as a geometrical measure which is capable of differentiating the segmented cell organelles. The trained model yields 96.52% accuracy with a loss of 0.07 for 50 epochs. Dice Index of 0.93, 0.76 and 0.75 is achieved for nuclei, ER and cytoplasm respectively. It is observed that nuclei to cytoplasm provides comparatively higher percentage change (74.56%) in the ratiometric index than nuclei to ER and ER to cytoplasm. The achieved results demonstrate that the proposed study can predict the cell painted organelles from a composite image with good performance measures. This study could be employed for generating cell painted organelles from raw microscopy images without using specific fluorescent labeling. [ABSTRACT FROM AUTHOR]

The systematic identification of tumour vulnerabilities through perturbational experiments on cancer models, including genome editing and drug screens, is playing a crucial role in combating cancer. This collective effort is known as the Cancer Dependency Map (DepMap). The 1st European Cancer Dependency Map Symposium (EuroDepMap), held in Milan last May, featured talks, a roundtable discussion, and a poster session, showcasing the latest discoveries and future challenges related to the DepMap. The symposium aimed to facilitate interactions among participants across Europe, encourage idea exchange with leading experts, and present their work and future projects. Importantly, it sparked discussions on future endeavours, such as screening more complex cancer models and accounting for tumour evolution. [ABSTRACT FROM AUTHOR]

Background: The tumor-adipose microenvironment (TAME) is characterized by the enrichment of adipocytes, and is considered a special ecosystem that supports cancer progression. However, the heterogeneity and diversity of adipocytes in TAME remains poorly understood. Methods: We conducted a single-cell RNA sequencing analysis of adipocytes in mouse and human white adipose tissue (WAT). We analyzed several adipocyte subtypes to evaluate their relationship and potential as prognostic factors for overall survival (OS). The potential drugs are screened by using bioinformatics methods. The tumor-promoting effects of a typical adipocyte subtype in breast cancer are validated by performing in vitro functional assays and immunohistochemistry (IHC) in clinical samples. Results: We profiled a comprehensive single-cell atlas of adipocyte in mouse and human WAT and described their characteristics, origins, development, functions and interactions with immune cells. Several cancer-associated adipocyte subtypes, namely DPP4+ adipocytes in visceral adipose and ADIPOQ+ adipocytes in subcutaneous adipose, are identified. We found that high levels of these subtypes are associated with unfavorable outcomes in four typical adipose-associated cancers. Some potential drugs including Trametinib, Selumetinib and Ulixertinib are discovered. Emphatically, knockdown of adiponectin receptor 1 (AdipoR1) and AdipoR2 impaired the proliferation and invasion of breast cancer cells. Patients with AdipoR2-high breast cancer display significantly shorter relapse-free survival (RFS) than those with AdipoR2-low breast cancer. Conclusion: Our results provide a novel understanding of TAME at the single-cell level. Based on our findings, several adipocyte subtypes have negative impact on prognosis. These cancer-associated adipocytes may serve as key prognostic predictor and potential targets for treatment in the future. [ABSTRACT FROM AUTHOR]

ARL-17477 is a selective neuronal nitric oxide synthase (NOS1) inhibitor that has been used in many preclinical studies since its initial discovery in the 1990s. In the present study, we demonstrate that ARL-17477 exhibits a NOS1-independent pharmacological activity that involves inhibition of the autophagy-lysosomal system and prevents cancer growth in vitro and in vivo. Initially, we screened a chemical compound library for potential anticancer agents, and identified ARL-17477 with micromolar anticancer activity against a wide spectrum of cancers, preferentially affecting cancer stem-like cells and KRAS-mutant cancer cells. Interestingly, ARL-17477 also affected NOS1-knockout cells, suggesting the existence of a NOS1-independent anticancer mechanism. Analysis of cell signals and death markers revealed that LC3B-II, p62, and GABARAP-II protein levels were significantly increased by ARL-17477. Furthermore, ARL-17477 had a chemical structure similar to that of chloroquine, suggesting the inhibition of autophagic flux at the level of lysosomal fusion as an underlying anticancer mechanism. Consistently, ARL-17477 induced lysosomal membrane permeabilization, impaired protein aggregate clearance, and activated transcription factor EB and lysosomal biogenesis. Furthermore, in vivo ARL-17477 inhibited the tumor growth of KRAS-mutant cancer. Thus, ARL-17477 is a dual inhibitor of NOS1 and the autophagy-lysosomal system that could potentially be used as a cancer therapeutic. [ABSTRACT FROM AUTHOR]

There is a long tradition in the Biophysics community of using simulations as a means to understand macromolecular behavior in various physicochemical methods. This allows a rigorous means to interpret observations in terms of fundamental principles, including chemical equilibrium, reaction kinetics, transport processes and thermodynamics. Here we simulate data for the Gilbert Theory for self-association, a fundamental analytical ultracentrifuge (AUC) technique to understand the shape of sedimentation velocity reaction boundaries that involve reversible monomer–Nmer interactions. Simulating monomer–dimer through monomer–hexamer systems as a function of concentration about the equilibrium constant allows a visual means to differentiate reaction stoichiometry by determining end points and inflection positions. Including intermediates (eg A1-A2-A3-A4-A5-A6) in the simulations reveals the smoothing of the reaction boundary and the removal of sharp inflections between monomers and polymers. The addition of cooperativity restores sharp boundaries or peaks to the observation and allows more discrimination in the selection of possible fitting models. Thermodynamic nonideality adds additional features when applied across wide ranges of concentration that might be appropriate for high-concentration therapeutic monoclonal antibody (mAb) solutions. This presentation serves as a tutorial for using modern AUC analysis software like SEDANAL for selecting potential fitting models. [ABSTRACT FROM AUTHOR]

Proper interpretation of analytical ultracentrifugation (AUC) data for purified proteins requires ancillary information and calculations to account for factors such as buoyancy, buffer viscosity, hydration, and temperature. The utility program SEDNTERP has been widely used by the AUC community for this purpose since its introduction in the mid-1990s. Recent extensions to this program (1) allow it to incorporate data from diffusion as well as AUC experiments; and (2) allow it to calculate the refractive index of buffer solutions (based on the solute composition of the buffer), as well as the specific refractive increment (dn/dc) of proteins based on their composition. These two extensions should be quite useful to the light scattering community as well as helpful for AUC users. The latest version also adds new terms to the partial specific volume calculations which should improve the accuracy, particularly for smaller proteins and peptides, and can calculate the viscosity of buffers containing heavy isotopes of water. It also uses newer, more accurate equations for the density of water and for the hydrodynamic properties of rods and disks. This article will summarize and review all the equations used in the current program version and the scientific background behind them. It will tabulate the values used to calculate the partial specific volume and dn/dc, as well as the polynomial coefficients used in calculating the buffer density and viscosity (most of which have not been previously published), as well as the new ones used in calculating the buffer refractive index. [ABSTRACT FROM AUTHOR]

Background: Skin cutaneous melanoma (SKCM) is an extremely malignant tumor that is associated with a poor prognosis. LSM2 has been found to be related to different types of tumors; however, its role in SKCM is poorly defined. We aimed to determine the value of LSM2 as a prognostic biomarker for SKCM. Methods: The expression profile of LSM2 mRNA was compared between tumor and normal tissues in public databases, such as TCGA, GEO, and BioGPS. LSM2 protein expression was explored using immunohistochemistry (IHC) on a tissue microarray containing 44 SKCM tissues and 8 normal samples collected at our center. Kaplan-Meier analysis was performed to assess the prognostic value of LSM2 expression in patients with SKCM. SKCM cell lines with LSM2 knockdown were used to determine the effects of LSM2. Cell counting kit-8 (CCK8) and colony formation assays were conducted to assess cell proliferation, whereas wound healing and transwell assays were carried out to assess the migration and invasion abilities of SKCM cells. Results: LSM2 was more highly expressed at the mRNA and protein levels in SKCM than that in normal skin. Moreover, elevated expression of LSM2 was associated with shorter survival time and early recurrence in patients with SKCM. The in vitro results revealed that the silencing of LSM2 in SKCM cells significantly inhibited cell proliferation, migration, and invasion. Conclusion: Overall, LSM2 contributes to malignant status and poor prognosis in patients with SKCM and may be identified as a novel prognostic biomarker and therapeutic target. [ABSTRACT FROM AUTHOR]

Evidence for a proposed activation mechanism is summarized. The low rate of ATP cleavage in the resting state of muscle is considered to result from the formation of a stable ring structure involving the two essential sulfhydryl groups on each myosin head and MgATP. Activation is thought to occur by interaction of actin in the vicinity of one of the essential sulfhydryl groups. Thus opening the stable ring leading to rapid dissociation of split products. This idea is consistent with the kinetic scheme of ATP cleavage developed recently by other workers and allows a prediction of the shift in population of intermediate states with changes in solvent conditions. It is also supported by our recent studies on the spatial geometry of the ring. The possibility that other nucleophilic groups may replace the sulfhydryl groups in other contractile systems is considered. The relevance of the ring structure to the tension generating event is discussed on the basis of recent measurements of the rate of contraction of modified (SH1-blocked) actomyosin threads. Results indicate the ability to form the ring structure is an essential requirement of the contractile process in these systems, and, moreover, that single, modified heads of myosin can act independently to produce the same rate of contraction as native myosin. This latter finding suggests that the myosin duplex exhibits some type of negative cooperativity in the contractile process.

The effect of divalent cations on the self-association of high molecular weight subfragment-2 (long S-2) and low molecular weight subfragment-2 (short S-2) of rabbit skeletal muscle myosin has been investigated. In the presence of millimolar concentrations of Ca2+ or Mg2+ long S-2 associates at neutral pH to form ordered, high molecular weight aggregates whereas short S-2 does not associate. The association process is co-operative and results from binding two to four divalent cations within the light meromyosin-heavy meromyosin (LMM-HMM) hinge region of long S-2. Optical diffraction of electron micrographs of the long S-2 aggregates revealed several periodicities including reflections near 143 A. High molecular weight HMM showed a similar divalent metal induced self-association. Chymotryptic digestion studies of rod filaments reveal that cleavage within the LMM-HMM hinge is also strongly dependent on the presence of divalent cations. At pH 8, in the absence of divalent cations, the S-2 region appears to be displaced away from the filament backbone resulting in rapid proteolysis in the hinge domain. At high cation concentrations (greater than 10 mM) proteolytic cleavage is suppressed. A similar depression of the (substantially lower) hinge cleavage rate was also observed at neutral pH following addition of these divalent metal ions. Results suggest that binding of Mg2+ within the hinge domain under physiological conditions may act to lock the cross-bridge onto the thick filament surface in its resting-state orientation.

Local melting within the subfragment-2 region of activated rabbit skeletal glycerinated muscle fibers has been investigated over the temperature range 5 to 37 degrees C, using an enzyme (chymotrypsin)-probe method. The cleavage rates were determined from the time-course of formation of digestion products by electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. We found the cleavage sites to be localized in a restricted region Mr = 64,000 to 90,000/polypeptide chain, measured from the C terminus of the myosin rod (the subfragment-2 hinge domain). The cleavage rate constant for activated muscle fibers in the presence of an ATP-regenerating system was about 100 times larger at each temperature than that for rigor or for relaxed muscle fibers and showed a marked increase in magnitude with increasing temperature. Comparative plots of the apparent rate-constant for cleavage within the subfragment-2 hinge domain and the isometric force generated by active fibers versus MgATP concentration gave closely similar profiles suggesting a strong positive correlation. Thus, there appears to be a close coupling between the conformational transition within the subfragment-2 hinge domain and contractile force when the cross-bridges undergo cycling.

The studies described in this paper focus on the structural stability of the S-2 segment of the myosin cross-bridge in rigor, relaxed and activated muscle. Enzyme-probe observations of myofibrils of rabbit psoas muscle in rigor reveal that the alpha-helical LMM/HMM hinge domain of S-2 undergoes substantial local melting near physiological temperatures when the S-2 portion of the cross-bridge is detached from the thick filament surface. This process is strongly suppressed under ionic conditions where the cross-bridge is bound to the filament backbone. Activation of glycerinated fiber bundles results in a dramatic increase (approximately 100 fold compared to rigor and relaxed fibers) in the rate of chymotryptic cleavage in the hinge domain consistent with an increase in local melting at several sites encompassing this region. Comparative plots of the apparent rate-constant for cleavage within the S-2 hinge and the isometric force generated by active fibers versus [MgATP] give similar profiles suggesting a close coupling between this conformational transition and contractile force. This interpretation appears to be in accord with recent laser T-jump experiments of rigor ("bridges up") and activated psoas muscle fibers which also suggest coupling between melting in S-2 and force generation.

The force-generating mechanism in muscle is discussed and it is shown that a helix-coil transition in the S-2 link of the cycling cross-bridge is compatible with the physical and chemical properties of this region of the myosin molecule. Thermal melting and temperature-jump experiments are described demonstrating that the light meromyosin-heavy meromyosin (LMM-HMM) hinge domain of S-2 is a segment of low thermal stability. This region can undergo alpha-helix-random coil transitions on a time-scale comparable to the quick-recovery tension transient observed when isometrically contracting muscle is abruptly shortened or stretched. Cross-linking and enzyme probe studies of glycerinated muscle fibres and myofibrils in resting, rigor and activating solvents suggest that the polypeptide chains within the hinge region of S-2 undergo a conformational transition to a more open, proteolytically sensitive structure when the S-2 link is released from the thick filament surface.

The radius of gyration and fraction helix as a function of temperature have been determined for myosin rod from four different species: rabbit, frog, scallop, and antarctic fish. Measurements from sodium dodecyl sulfate gel electrophoresis indicate that all particles have the same molecular weight (approximately 130K). All fragments are nearly 100% alpha-helical at low temperatures (0-5 degrees C). The melting profiles for each are qualitatively similar in shape, but their midpoints are shifted along the temperature axis in the following order: antarctic fish (Tm = 33 degrees C), scallop (Tm = 39 degrees C), frog (Tm = 45 degrees C), and rabbit (Tm = 49 degrees C). Corresponding radius of gyration vs temperature profiles for each species are shifted to lower temperatures (approximately 5-8 degrees C) with respect to the optical rotation melting curves. From plots of radius of gyration vs fraction helix, we find a marked drop in the radius of gyration (from 43 to approximately 34 nm) with less than a 5% decrease in fraction helix for rabbit, frog, and antarctic fish rods, whereas the radius of gyration of scallop rod never exceeds 34 nm. Results indicate hinging of the myosin rod of each species. The thermal stabilities of the myosin rods shift in parallel with the working temperature of their respective muscles.

We have examined the effect of cross-linking on cross-bridge movement and isometric force in glycerinated psoas fibers. Two different methods, high-porosity gel electrophoresis and a fractionation technique, were used to follow the cross-linking of myosin heads (subfragment 1) and rod segments to the thick filament backbone. Contrary to earlier reports [Sutoh, K., & Harrington, W. F. (1977) Biochemistry 16, 2441-2449; Sutoh, K., Chiao, Y. C., & Harrington, W. F. (1978) Biochemistry 17, 1234-1239; Chiao, Y. C., & Harrington, W. F. (1979) Biochemistry 18, 959-963], we find that the heads of the myosin molecules are not cross-linked to the thick filament surface by dimethyl suberimidate. The time dependence of cross-linking rod segments within the core was monitored by a disulfide oxidation procedure to distinguish between intermolecular and intramolecular cross-linking. Comparison of the extent of the cross-linking reaction within myofibrils and the isometric force developed within fibers at various stages of cross-linking shows that isometric force is abolished in parallel with the formation of high molecular weight (cross-linked) rod species (greater than or equal to Mr 1000K). The myofibrillar ATPase remains virtually unaffected by the cross-linking reaction.

Temperature-jump studies on the long S-2 fragment (100,000 daltons) isolated from myosin show that this structure can undergo alpha-helix--random coil transitions in a time range approximating the cycle time of a crossbridge. Two relaxation times are observed after temperature jumps of 5 degrees C over the range 35--55 degrees C, one in the submillisecond (tau f) and the other in the millisecond (tau s) time ranges. Both processes exhibit maxima near the midpoint of the helix--coil transition (tm = 45 +/- 2 degrees C) as determined by optical rotation melt experiments. Similar results were observed for the low temperature transition (tm = 45 degrees C) of the myosin rod. Viscosity studies reveal that the S-2 particles has significant flexibility at physiological temperature. Results are considered in terms of the Huxley--Simmons and helix--coil transition models for force generation in muscle.

An iodine photodissociation laser generates the 1.315-micron infrared light used to heat the fiber and solvent. Heating of the cell contents by the direct absorption of laser energy is complete within the 100-microseconds rise time of the force transducer. A 5 degrees C temperature jump was usual. Interference with the force record by shock waves, electromagnetic disturbances, and uneven heating of fiber and solvent is minimal and close to the normal background noise of the transducer output. The postjump temperature of the cell remains static for a minimum of 400 ms before evidence of cooling is seen. The temperature of the cell could be changed rapidly. The cuvette contents could therefore be rapidly raised to, and lowered from, elevated prejump temperatures. As a result, sensitive biological samples are subjected to potentially denaturing conditions for the minimum length of time required for the temperature jump. Experiments on collagen and muscle fibers in which normal and rubber-like thermoelastic responses are kinetically resolved from each other are presented. The instrument offers substantial improvements in performance over other currently available designs.

A clear prediction of the helix-coil model for force generation in muscle is that force should be produced when the equilibrium (helix-coil) of a rigor (or activated) fiber is perturbed by a temperature jump near the melting temperature of the light meromyosin/heavy meromyosin hinge. An infrared, iodine-photodissociation laser was used to heat the fibers by approximately equal to 5 degrees C in under 1 mus. Under ionic conditions where rigor bridges are predominantly associated with the thick filament backbone, an abrupt drop in tension typical of normal thermoelastic expansion was seen. A similar response was observed below 41 degrees C for thick filament-released rigor bridges. Above this temperature, a rubber-like thermoelastic response was obtained typical of a helix-coil transition. At temperatures near 50 degrees C, the amount of force generated by a rigor fiber was large and comparable to that seen for an activated fiber at 5 degrees C. The relaxation spectra of force generation obtained for both systems (rigor and activated) show a step change followed by a biexponential kinetic process. The reciprocal relaxation times and amplitudes for these individual processes in activated and rigor fibers differ only by factors of 2-4. Force generation in the rigor muscle appears to arise from melting in the subfragment 2 hinge region of the myosin molecule since binding of subfragment 2 to the thick filament backbone inhibits force production. No significant force generation was observed following temperature jumps of relaxed fibers.

Effects of temperature and pH on intact rabbit and chicken myosin, isolated myosin rods, rabbit subfragment-2 (61 kDa, 53 kDa, and 34 kDa) and chicken light meromyosin (LMM) fragments were tested to induce a phase transition from alpha-helix to coil conformation, within the hinge region. The influence of temperature and pH were studied directly with length determination by electron microscopy. An increase of temperature to 50 degrees C yielded a shortening of 16 nm, 8 to 9 nm and 7 to 11 nm for intact myosin, isolated rods and long S-2 fragments, respectively. The length of the 34 kDa short S-2 and LMM fragments were unchanged. An increase of pH from neutral to pH 8.0 yielded values that were somewhat smaller, e.g. 12 nm, 6 nm and 6 to 8 nm for intact myosin, isolated rods and long S-2 fragments, respectively, whereas the 34 kDa short S-2 LMM fragments were also unaffected. Thus, melting and subsequent shortening is confined to the region between LMM and short S-2 segment, that is the hinge region. Alteration of temperature had a stronger shortening effect than alteration of pH, and shortening of long S-2 was more pronounced under physiological salt conditions as compared with high (0.3 M) salt. The shortening of rods in intact myosin amounted to twice the value observed with isolated rods. The amount of contraction was somewhat smaller in rods than in the 61 kDa and 53 kDa long S-2 fragments.

14C-Labeled fluorodinitrobenzene and N-ethylmaleimide have been used as chemical probes of the conformational states of myosin induced by the binding of MgADP and MgATP. The results indicate that in the high-energy conformation, MMgADP-Pi, the essential thiols are protected from modification but their diminished reactivity does not result from depletion of the reagent by reaction at nonessential thiols. The binding of MgADP to myosin exposes the essential thiols as reflected by an increased rate of their modification. The influence of the divalent cations Mg2+ and Ca2+ on the conformation of the M species has also been investigated. By monitoring the incorporation of fluorodinitrobenzene, the conformations of the M state in the presence of these cations can be clearly discerned.

Studies were conducted on the physical properties of Enzyme I, the first protein in the Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system. Since values lower than those previously reported for the monomer molecular weight were obtained, experiments were performed to determine whether Enzyme I had been partially degraded during isolation of homogeneous protein. Crude extracts and partially purified and homogeneous protein preparations exhibited identical behavior in crossed immunoelectrophoresis analyses, indicating that the isolated protein represented native, intact Enzyme I. The monomeric subunit of Enzyme I is globular, with a frictional ratio of about 1. Sedimentation equilibrium experiments provided a monomer molecular weight of 57,700 +/- 3,400, and gel filtration studies under denaturing conditions gave a comparable value of 57,000. The values previously obtained from polyacrylamide gel electrophoresis analyses in the presence of sodium dodecyl sulfate varied with the conditions used, but under one set of conditions agreed with those given above. The sedimentation equilibrium studies were conducted at 8 degrees C, in the absence of substrates and cofactor (phosphoenolpyruvate, pyruvate, Mg2+). Under these conditions Enzyme I self-associates, but the association is weak, favoring primarily monomer. Because of solubility limitations, the sedimentation experiments were performed with Enzyme I at an initial concentration of 0.5 mg/ml, providing a concentration distribution of 0.1 to 2 mg/ml. Computer analysis of the results showed that within this concentration range it was not possible to distinguish between two modes of self-association, monomer-dimer and isodesmic. The physiological significance of the results is discussed.

The thermal stability and melting kinetics of the alpha-helical conformation within several regions of the rabbit myosin rod have been investigated. Cyanogen bromide cleavage of long myosin subfragment-2 produced one coiled-coil alpha-helical fragment corresponding to short subfragment-2 with molecular weight 90,000 (Mr = 45,000) and two fragments from the hinge region with molecular weights of 32,000 to 34,000 (Mr = 16,000 to 17,000) and 24,000 to 26,000 (Mr = 12,000 to 13,000). Optical rotation melting experiments and temperature-jump kinetic studies of long subfragment-2 and its cyanogen bromide fragments show that the hinge and the short subfragment-2 domains melt as quasi-independent co-operative units. The alpha-helical structure within the hinge has an appreciably lower thermal stability than the flanking short subfragment-2 and light meromyosin regions of the myosin rod. Two relaxation processes for helix-melting, one in the submillisecond range (tau f) and the other in the millisecond range (tau s), are observed in the light meromyosin and short subfragment-2 regions of the rod, but melting in the hinge domain is dominated by the fast (tau f) process. Results suggest that the hinge domain of the subfragment-2 link may be the locus of force generation in a cycling cross-bridge.

Synthetic thick filaments were cross-linked with dimethyl suberimidate at various pH values over the range pH 6.8---8.3. The rate of cross-linking myosin heads to the thick filament surface decreases significantly over a narrow pH range (7.4--8.0) despite the fact that the rate of the chemical reaction (amidination of lysine side chains) shows a positive pH dependence. The fall in rate cannot be ascribed to dissociation of the filament during the cross-linking reaction since the sedimentation boundary of the cross-linked filament (pH 8.3) remains unaltered in the presence of high salt (0.5 M). The decreased rate of cross-linking is also not caused by a shift in reactivity of a small number of highly reactive lysine groups, since the time course of cross-linking (pH 7.2) is unaffected by preincubation with a monofunctional imidate ester. Our results suggest that the heads of the myosin molecules move away from the thick filament surface at alkaline pH but are held close to the surface at neutral pH.

We have determined the rates of chymotryptic proteolysis of the myosin hinge region in glycerinated rabbit psoas fibers and myofibrils in in rigor-inducing, activating, and relaxing buffers. The time course of formation of light meromyosin (LMM) provides a specific probe for cleavage within the hinge domain. In rigor-inducing and relaxing buffers proteolysis within the hinge is depressed, but on activation LMM is formed at a markedly increased rate, which is dependent on the concentration of MgATP. Peptide bond cleavage occurs at four widely separated sites spanning the length of the hinge domain. Only a trivial amount of proteolysis occurs at the head--rod swivel or within the heavy chain of the head itself (S-1 subunit) in rigor-inducing and relaxing solvents, and we find no significant change on activation. The rate of formation of LMM in rigor-inducing buffer is unchanged by addition of MgADP, Pi, or magnesium adenosine 5'-[beta, gamma-imido]triphosphate or in activating solvent at zero overlap between thick and thin filaments. These results provide evidence for a conformational (helix--coil) transition within the myosin hinge upon activation of skeletal muscle.

Analysis of the early tension responses after abrupt step changes in the length of isometrically contracting skeletal muscle shows that the magnitude of the recovery tension (T2) and the time dependence of this process at various step displacements give a good correlation with the behavior expected for a helix-coil transition in the subfragment-2 (S-2) region of myosin. The "instantaneous" tension response (to T1) after the step change in length appears to have its origin in compliance within the coil region of S-2, which is formed through helix melting at the moment of force generation.