Identification and validation of novel and more effective choline kinase inhibitors against Streptococcus pneumoniae.

Streptococcus pneumoniae choline kinase (sChoK) has previously been proposed as a drug target, yet the effectiveness of the first and only known inhibitor of sChoK, HC-3, is in the millimolar range. The aim of this study was thus to further validate sChoK as a potential therapeutic target by discovering more powerful sChoK inhibitors. LDH/PK and colorimetric enzymatic assays revealed two promising sChoK inhibitor leads RSM-932A and MN58b that were discovered with IC50 of 0.5 and 150 µM, respectively, and were shown to be 2-4 magnitudes more potent than the previously discovered inhibitor HC-3. Culture assays showed that the minimum inhibitory concentration (MIC) of RSM-932A and MN58b for S. pneumoniae was 0.4 µM and 10 µM, respectively, and the minimum lethal concentration (MLC) was 1.6 µM and 20 µM, respectively. Western blot monitoring of teichoic acid production revealed differential patterns in response to each inhibitor. In addition, both inhibitors possessed a bacteriostatic mechanism of action, and neither interfered with the autolytic effects of vancomycin. Cells treated with MN58b but not RSM-932A were more sensitive to a phosphate induced autolysis with respect to the untreated cells. SEM studies revealed that MN58b distorted the cell wall, a result consistent with the apparent teichoic acid changes. Two novel and more highly potent putative inhibitors of sChoK, MN58b and RSM-932A, were characterized in this study. However, the effects of sChoK inhibitors can vary at the cellular level. sChoK inhibition is a promising avenue to follow in the development of therapeutics for treatment of S. pneumoniae.

Metabolomic and Imaging Mass Spectrometric Assays of Labile Brain Metabolites: Critical Importance of Brain Harvest Procedures.

Metabolomic technologies including imaging mass spectrometry (IMS; also called mass spectrometry imaging, MSI, or matrix-assisted laser desorption/ionization-mass spectrometry imaging, MALDI MSI) are important methods to evaluate levels of many compounds in brain with high spatial resolution, characterize metabolic phenotypes of brain disorders, and identify disease biomarkers. ATP is central to brain energetics, and reports of its heterogeneous distribution in brain and regional differences in ATP/ADP ratios reported in IMS studies conflict with earlier studies. These discordant data were, therefore, analyzed and compared with biochemical literature that used rigorous methods to preserve labile metabolites. Unequal, very low regional ATP levels and low ATP/ADP ratios are explained by rapid metabolism during postmortem ischemia. A critical aspect of any analysis of brain components is their stability during and after tissue harvest so measured concentrations closely approximate their physiological levels in vivo. Unfortunately, the requirement for inactivation of brain enzymes by freezing or heating is not widely recognized outside the neurochemistry discipline, and procedures that do not prevent postmortem autolysis, including decapitation, brain removal/dissection, and 'snap freezing' are commonly used. Strong emphasis is placed on use of supplementary approaches to calibrate metabolite abundance in units of concentration in IMS studies and comparison of IMS results with biochemical data obtained by different methods to help identify potential artifacts.

Dissecting capture and twisting of aureolysin and pseudolysin: functional amino acids of the Dispase autolysis-inducing protein.

The Dispase autolysis-inducing protein (DAIP) from Streptomyces mobaraensis attracts M4 metalloproteases, which results in inhibition and autolysis of bacillolysin (BL) and thermolysin (TL). The present study shows that aureolysin (AL) from Staphylococcus aureus and pseudolysin (LasB) from Pseudomonas aeruginosa are likewise impaired by DAIP. Complete inhibition occurred when DAIP significantly exceeded the amount of the target protease. At low DAIP concentrations, AL and BL performed autolysis, while LasB and TL degradation required reductants or detergents that break intramolecular disulfide bonds or change the protein structure. Site directed mutagenesis of DAIP and removal of an exposed protein loop either influenced binding or inhibition of AL and TL but had no effect on LasB and BL. The Y170A and Δ239-248 variants had completely lost affinity for TL and AL. The exchange of Asn-275 also impaired the interaction of DAIP with AL. In contrast, DAIP Phe-297 substitution abolished inhibition and autolysis of both target proteases but still allowed complex formation. Our results give rise to the conclusion that other, yet unknown DAIP amino acids inactivate LasB and BL. Obviously, various bacteria in the same habitat caused Streptomyces mobaraensis to continuously optimize DAIP in inactivating the tackling metalloproteases.

Immunohistochemical Study of Dark and Progenitor Carotid Body Cells: Artefacts or Real Subtypes?

Postmortem changes occurring in human carotid body were simulated on the Wistar rat model. It was shown that light, dark, and pyknotic (progenitor) subtypes of human carotid body cells are an artifact and cannot be used in clinical practice to study the characteristics of various human diseases. The differences between the control group of healthy individuals and individuals with the various pathologies are most likely due to the different levels of premortal hypoxia that the tissue had been exposed to. Moreover, widespread antigens used in practice were divided into 2 groups by their tolerance to autolysis: stable and unstable ones. This can be useful for the development of immunohistochemical test algorithms for the diagnostics on autopsy material.

Mitochondrial apoptosis and proteolytic changes of myofibrillar proteins in two different pork muscles during aging.

This study aims to investigate the changes in mitochondrial apoptotic factors and proteolysis of two porcine muscles (psoas major - PM and longissimus dorsi - LD) during aging. Results found that during 2-168 h postmortem mitochondrial membrane permeability, mitochondrial lipid peroxidation, Ca2+ levels were increased, while the reduction level and abundance of cytochrome c were decreased (P < 0.05) in both muscle types. Furthermore, the activation of caspase-3 along with increases in troponin-T and desmin degradation, and µ-calpain autolysis were found (P < 0.05), regardless of muscle type. PM maintained higher mitochondrial apoptotic factors, but had more intact desmin, less troponin-T degradation and less extent of autolyzed products of µ-calpain compared to LD (P < 0.05). These results indicate that the rapid onset of mitochondrial apoptosis of PM would not lead to a subsequent impact on myofibrillar protein degradation, suggesting that the mitochondrial apoptosis mediated tenderization process could be muscle-specific.

Effect of nitric oxide and calpastatin on the inhibition of µ-calpain activity, autolysis and proteolysis of myofibrillar proteins.

The aim of this study was to investigate the dual effect of the nitric oxide donor NOR-3 and calpastatin on µ-calpain activity, autolysis, and proteolytic ability. µ-Calpain and calpastatin were purified and allocated to the following five treatments: µ-calpain, µ-calpain + calpastatin, µ-calpain + NOR-3, µ-calpain + calpastatin + NOR-3, and µ-calpain + NOR-3 + calpastatin. µ-Calpain autolysis and the activity against purified myofibrils was initiated by addition of calcium. Results showed that NOR-3 could induce µ-calpain S-nitrosylation and effectively block the activity via the inhibition of µ-calpain autolysis. Calpastatin inhibited µ-calpain activity in a dose-dependent manner. The combined treatment of NOR-3 and calpastatin exerted a further inhibitory effect on µ-calpain activity, autolysis and proteolysis which was affected by the addition order of NOR-3 and calpastatin. Our data suggest that S-nitrosylation may play a regulatory role in mediating µ-calpain activity in the presence of calpastatin.

Pneumococcal DNA-binding proteins released through autolysis induce the production of proinflammatory cytokines via toll-like receptor 4.

Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis. We demonstrated that recombinant (r) DnaK, rEF-Tu, and rGAPDH induced significantly higher levels of interleukin-6 and tumor necrosis factor production in peritoneal macrophages and THP-1-derived macrophage-like cells via toll-like receptor 4. Furthermore, the DNA-binding activity of these proteins was confirmed by surface plasmon resonance assay. We demonstrated that pneumococcal DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in macrophages, and might cause host tissue damage and affect the development of pneumococcal diseases.

Increased Autolysis of µ-Calpain in Skeletal Muscles of Chronic Alcohol-Fed Rats.

BACKGROUND: Proteolysis can proceed via several distinct pathways such as the lysosomal, calcium-dependent, and ubiquitin-proteasome-dependent pathways. Calpains are the main proteases that cleave a large variety of proteins, including the giant sarcomeric proteins, titin and nebulin. Chronic ethanol feeding for 6 weeks did not affect the activities of µ-calpain and m-calpain in the m. gastrocnemius. In our research, changes in µ-calpain activity were studied in the m. gastrocnemius and m. soleus of chronically alcohol-fed rats after 6 months of alcohol intake. METHODS: SDS-PAGE analysis was applied to detect changes in titin and nebulin contents. Titin phosphorylation analysis was performed using the fluorescent dye Pro-Q Diamond. Western blotting was used to determine µ-calpain autolysis as well as µ-calpain and calpastatin contents. The titin and nebulin mRNA levels were assessed by real-time PCR. RESULTS: The amounts of the autolysed isoform (78 kDa) of full-length µ-calpain (80 kDa) increased in the m. gastrocnemius and m. soleus of alcohol-fed rats. The calpastatin content increased in m. gastrocnemius. Decreased intact titin-1 (T1) and increased T2-proteolytic fragment contents were found in the m. gastrocnemius and m. soleus of the alcohol-fed rats. The nebulin content decreased in the rat gastrocnemius muscle of the alcohol-fed group. The phosphorylation levels of T1 and T2 were increased in the m. gastrocnemius and m. soleus, and decreased titin and nebulin mRNA levels were observed in the m. gastrocnemius. The nebulin mRNA level was increased in the soleus muscle of the alcohol-fed rats. CONCLUSIONS: In summary, our data suggest that prolonged chronic alcohol consumption for 6 months resulted in increased autolysis of µ-calpain in rat skeletal muscles. These changes were accompanied by reduced titin and nebulin contents, titin hyperphosphorylation, and development of hindlimb muscle atrophy in the alcohol-fed rats.

Post Mortem Redistribution of Drugs: Current State of Knowledge.

BACKGROUND: Drug concentrations obtained from post mortem samples do not necessarily reflect the concentrations at the time of death, and variations of concentration may be observed between different sites and/or different sampling times. These phenomena, collectively termed post mortem redistribution, concern numerous molecules (medications, drugs of abuse, gases, etc.) and can complicate the interpretation of toxicological analyses. METHODS: Literature review. RESULTS: The mechanisms that cause these phenomena are complex and often intricate. Certain organs, which concentrate the molecules before death, may release them very early in the vascular sector. The gastrointestinal tract, liver, lungs and myocardium are mainly concerned. Cell autolysis also plays a part in drug release. Furthermore, micro-organisms (mainly bacteria and yeasts) which colonize the organism during putrefaction may cause neoformation and/or the degradation of certain molecules. Lastly, it appears that the physicochemical and pharmacokinetic profile of xenobiotics, notably their lipophilic nature, their ionization state and their volume of distribution may be factors likely to influence redistribution phenomena. Some recommendations concerning anatomic sampling sites, sampling methods and sample storage make it possible to limit these phenomena.

Function of bacterial community dynamics in the formation of cadaveric semiochemicals during in situ carcass decomposition.

The decomposition of dead mammalian tissue involves a complex temporal succession of epinecrotic bacteria. Microbial activity may release different cadaveric volatile organic compounds which in turn attract other key players of carcass decomposition such as scavenger insects. To elucidate the dynamics and potential functions of epinecrotic bacteria on carcasses, we monitored bacterial communities developing on still-born piglets incubated in different forest ecosystems by combining high-throughput Illumina 16S rRNA sequencing with gas chromatography-mass spectrometry of volatiles. Our results show that the community structure of epinecrotic bacteria and the types of cadaveric volatile compounds released over the time course of decomposition are driven by deterministic rather than stochastic processes. Individual cadaveric volatile organic compounds were correlated with specific taxa during the first stages of decomposition which are dominated by bacteria. Through best-fitting multiple linear regression models, the synthesis of acetic acid, indole and phenol could be linked to the activity of Enterobacteriaceae, Tissierellaceae and Xanthomonadaceae, respectively. These conclusions are also commensurate with the metabolism described for the dominant taxa identified for these families. The predictable nature of in situ synthesis of cadaveric volatile organic compounds by epinecrotic bacteria provides a new basis for future chemical ecology and forensic studies.

Effect of protein S-nitrosylation on autolysis and catalytic ability of µ-calpain.

The effect of S-nitrosylation on the autolysis and catalytic ability of µ-calpain in vitro in the presence of 50µM Ca(2 +) was investigated. µ-Calpain was incubated with different concentrations of nitric oxide donor S-nitrosoglutathione (GSNO) and subsequently reacted with purified myofibrils. Results showed that the amount of 80kDa µ-calpain subunit significantly decreased as GSNO increased from 0 to 300µM, but increases of GSNO to 300, 500 and 1000µM did not result in further inhibition. The catalytic ability of nitrosylated µ-calpain to degrade titin, nebulin, troponin-T and desmin was significantly reduced when the GSNO concentration was higher than 300µM. The cysteine residues of µ-calpain at positions 49, 351, 384, and 592 in the catalytic subunit and at 142 in small subunit were S-nitrosylated, which could be responsible for decreased µ-calpain activity. Thus, S-nitrosylation can negatively regulate the activation of µ-calpain resulting in decreased proteolytic ability on myofibrils.

D-Alanine-Controlled Transient Intestinal Mono-Colonization with Non-Laboratory-Adapted Commensal E. coli Strain HS.

Soon after birth the mammalian gut microbiota forms a permanent and collectively highly resilient consortium. There is currently no robust method for re-deriving an already microbially colonized individual again-germ-free. We previously developed the in vivo growth-incompetent E. coli K-12 strain HA107 that is auxotrophic for the peptidoglycan components D-alanine (D-Ala) and meso-diaminopimelic acid (Dap) and can be used to transiently associate germ-free animals with live bacteria, without permanent loss of germ-free status. Here we describe the translation of this experimental model from the laboratory-adapted E. coli K-12 prototype to the better gut-adapted commensal strain E. coli HS. In this genetic background it was necessary to complete the D-Ala auxotrophy phenotype by additional knockout of the hypothetical third alanine racemase metC. Cells of the resulting fully auxotrophic strain assembled a peptidoglycan cell wall of normal composition, as long as provided with D-Ala and Dap in the medium, but could not proliferate a single time after D-Ala/Dap removal. Yet, unsupplemented bacteria remained active and were able to complete their cell cycle with fully sustained motility until immediately before autolytic death. Also in vivo, the transiently colonizing bacteria retained their ability to stimulate a live-bacteria-specific intestinal Immunoglobulin (Ig)A response. Full D-Ala auxotrophy enabled rapid recovery to again-germ-free status. E. coli HS has emerged from human studies and genomic analyses as a paradigm of benign intestinal commensal E. coli strains. Its reversibly colonizing derivative may provide a versatile research tool for mucosal bacterial conditioning or compound delivery without permanent colonization.

A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome.

Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.

Evaluation of Pseudotrypsin Cleavage Specificity Towards Proteins by MALDI-TOF Mass Spectrometry.

Trypsin is a protease, which is commonly used for the digestion of protein samples in proteomic experiments. The process of trypsin autolysis is known to produce autolytic peptides as well as active enzyme forms with one or more intra-chain splits. In consequence, their variable presence can influence the digestion of a protein substrate in the reaction mixture. Besides two major and well-studied forms named ß-trypsin and α-trypsin, there are also other active trypsin forms known such as γ-trypsin and pseudotrypsin (ψ-trypsin). In this work, the cleavage specificity of ψ-trypsin was evaluated using in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses of the resulting peptides. The numbers of produced and matching peptides were similar to those obtained using α-/ß-trypsin. The same experience was obtained with a real complex protein sample from rat urine. In previous reports, ψ-trypsin was supposed to generate non-specific cleavages, which has now been reevaluated. Purified ψ-trypsin cleaved all analyzed proteins preferentially on the C-terminal side of Lys and Arg residues in accordance with the canonical tryptic cleavage. However, a minor nonspecific cleavage performance was also registered (particularly after Tyr and Phe), which was considerably higher than in the case of trypsin itself.

The carbon starvation response of the ectomycorrhizal fungus Paxillus involutus.

The amounts of carbon allocated to the fungal partner in ectomycorrhizal associations can vary substantially depending on the plant growth and the soil nutrient conditions, and the fungus may frequently be confronted with limitations in carbon. We used chemical analysis and transcriptome profiling to examine the physiological response of the ectomycorrhizal fungus Paxillus involutus to carbon starvation during axenic cultivation. Carbon starvation induced a decrease in the biomass. Concomitantly, ammonium, cell wall material (chitin) and proteolytic enzymes were released into the medium, which suggest autolysis. Compared with the transcriptome of actively growing hyphae, about 45% of the transcripts analyzed were differentially regulated during C-starvation. Induced during starvation were transcripts encoding extracellular enzymes such as peptidases, chitinases and laccases. In parallel, transcripts of N-transporters were upregulated, which suggest that some of the released nitrogen compounds were re-assimilated by the mycelium. The observed changes suggest that the carbon starvation response in P. involutus is associated with complex cellular changes that involves autolysis, recycling of intracellular compounds by autophagy and reabsorption of the extracellular released material. The study provides molecular markers that can be used to examine the role of autolysis for the turnover and survival of the ectomycorrhizal mycelium in soils.

Markers of Autolysis in Acute ST Elevation Myocardial Infarction.

INTRODUCTION: The availability of reliable noninvasive markers for infarct-related artery (IRA) patency status are very limited, otherwise could allow early identification of patients with patent IRA, for whom repeat thrombolysis or rescue percutaneous coronary intervention (PCI) are not necessary. METHODS: We conducted a single centered retrospective study of STEMI patients undergoing primary PCI to determine how various factors such as demographic characteristics, risk markers of coronary heart disease, clinical and blood parameters present differently in patients with higher coronary flow and patent infarct related artery from patients with total occlusion at the time of initial angiography and how they affect in outcome of the disease. RESULTS: MPV level (11.96 fL vs. 10.92 fL, P < 0.001), Lp (a) level (179.57 nmol/l vs 141.16 nmol/l , p < 0.001), CK-MB (290.2 vs. 190.98, P < 0.001), total cholesterol level (4.11 mmol/L vs. 3.8 mmol/L, p < 0.02) in total occlusion group were higher than in the patent IRA group. Wall motion abnormality was 77.2% for 203 patients with total occlusion group and 54.2% for 83 patients with patent IRA group (P<0.01). Mean hospital stay days were higher in total occlusion group as compared to the patent IRA group P < 0.01. CONCLUSIONS: MVP, Lp (a), TC, and CK-MB levels and myocardial wall motion at the presentation may play the role of markers for IRA patency status that will help in early identification of patients with IRA, for whom repeat thrombolysis or rescue PCI may not be required.

The development of meat tenderness is likely to be compartmentalised by ultimate pH.

Bull Musculus longissimus dorsi (n=63) were categorised into high (pH≥6.2), intermediate (pH 5.8-6.19) and low (≤5.79) ultimate pH (pHu) and aged up to 28 days post mortem at -1°C. High pHu samples were acceptably tender at 1 day post mortem and significantly more tender than low pHu meat at all ageing timepoints (p<0.05). Rapid autolysis of µ-calpain in high pHu meat was linked with the more rapid degradation of titin, nebulin and filamin in this pHu group. Desmin degraded faster in low pHu meat and was concurrent with an increase of cathepsin B levels. The results from this study support the hypothesis that beef tenderisation is pHu compartmentalised with tenderness in high and low pHu meat characterised by variable rate of degradation of high and low molecular weight myofibrillar proteins during ageing, which are in turn regulated by µ-calpain and cathepsin B activities.

Structural studies of the cell wall polysaccharides from three strains of Lactobacillus helveticus with different autolytic properties: DPC4571, BROI, and LH1.

Lactobacillus helveticus is traditionally used in dairy industry as a starter or an adjunct culture for manufacture of cheese and some types of fermented milk. Its autolysis releases intracellular enzymes which is a prerequisite for optimum cheese maturation, and is known to be strain dependent. Autolysis is caused by an enzymatic hydrolysis of the cell wall peptidoglycan (PG) by endogenous peptidoglycan hydrolases (PGHs) or autolysins. Origins of differences in autolytic properties of different strains are not fully elucidated. Regulation of autolysis possibly depends on the structure of the cell wall components other than PG, particularly polysaccharides. In the present work, we screened six L. helveticus strains with different autolytic properties: DPC4571, BROI and LH1. We established, for the first time, that cell walls (CWs) of these strains contained polysaccharides, different from their CW teichoic acids. Cell wall polysaccharides of three strains were purified, and their chemical structures were established by 2D NMR spectroscopy and methylation analysis. The structures of their repeating units are presented.

Purification and autolysis of the ficin isoforms from fig (Ficus carica cv. Sabz) latex.

Ficin (EC 3.4.22.3), a cysteine endoproteolytic protease in fig trees' latex, has multiple isoforms. Until now, no data on autolysis of individual ficins (ficin isoforms) are available. Following purification, ficins' autolysis was determined by HPLC chromatogram changes and ultrafiltrations at different temperatures and storage times. These results showed that the number of HPLC peaks in latex proteins purification of Ficus carica cv. Sabz varied from previous fig varieties or cultivars. Proteolytic activity of ficins was inhibited by specific cysteine protease inhibitors, confirming the participation of the cysteine residue in the active site. The zeta potential of the first two eluted peaks (I and II) was negative, while that of other peaks were positive. All ficins were susceptible to autolysis when stored at high temperatures. In contrast, only the last two ficins (B, C) were prone to autolysis at cold temperature after long storage period. The rate of degradation of the ficins was significantly increased with the increased storage time. The ficin (A) related to peak (III) had the highest and the lowest surface hydrophobic patches and ratio of autolytic to proteolytic activity, respectively.

Activation of skeletal muscle calpain-3 by eccentric exercise in humans does not result in its translocation to the nucleus or cytosol.

The skeletal muscle-specific calpain-3 protease is likely involved in muscle repair, although the mechanism is not known. Physiological activation of calpain-3 occurs 24 h following eccentric exercise in humans. Functional consequences of calpain-3 activation are not known; however, calpain-3 has been suggested to be involved in nuclear signaling via NF-κB. To test this and help identify how/where calpain-3 acts, we investigated whether calpain-3 autolysis (hence, activation) following eccentric exercise results in translocation from its normal myofibrillar location to the nucleus or the cytosol. In resting human skeletal muscle, the majority (87%) of calpain-3 was present in myofibrillar fractions, with only a small proportion (<10%) in an autolyzed state. Enriched nuclear fractions contained ∼8% of the total calpain-3, which was present in a predominantly (>80%) autolyzed state. Using freshly dissected human muscle fibers to identify freely diffusible proteins, we showed that only ∼5% of the total calpain-3 pool was cytosolic. At 3 and 24 h following eccentric step exercise, there was an ∼70% increase in autolysis in whole muscle samples (n = 11, P < 0.05, by 1-way ANOVA with repeated measures and Newman-Keuls post hoc analysis). This exercise-induced autolysis was attributed to myofibrillar-bound calpain-3, since neither the amount of calpain-3 nor the proportion autolyzed was significantly changed in enriched nuclear or cytosolic fractions following the exercise intervention. We present a model for calpain-3 localization at rest and following activation in human skeletal muscle and suggest that the functional importance of calpain-3 remains predominantly tightly associated with its localization within the myofibrillar compartment.