PAM/protein-associated motor complex

For substrate input, MB-10 (100 μM) is a protein input diluent [1]. Protein input substrate input diluent MB-10 (0-100 μM) is used [1]. Protein input substrate input diluent MB-10 (0-100 μM) is used [1].
Small molecule mitochondrial import blockers of the Carla Koehler laboratory (MB)-10 inhibited import of substrates that require the TIM23 translocon. Mutational analysis coupled with molecular docking and molecular dynamics modeling revealed that MB-10 binds to a specific pocket in the C-terminal domain of Tim44 of the protein-associated motor (PAM) complex. This region was proposed to anchor Tim44 to the membrane, but biochemical studies with MB-10 show that this region is required for binding to the translocating precursor and binding to mtHsp70 in low ATP conditions. This study also supports a direct role for the PAM complex in the import of substrates that are laterally sorted to the inner membrane, as well as the mitochondrial matrix. Thus, MB-10 is the first small molecule modulator to attenuate PAM complex activity, likely through binding to the C-terminal region of Tim44.[1]
MB-10 is a potential attenuator of protein import into mitochondria. MB-10 Targets the TIM23 Translocon. MB-10 inhibits the import of substrates that use the TIM23 import pathway. MB-10 inhibition of import depends on specific chemical characteristics.  MB-10 Impairs the Import of Precursors Sorted to the Matrix and the Intermembrane Space. Specific Properties of MB-10 Inhibit Protein Import. MB-10 Targets Tim44 of the PAM Complex. Mutations in the α4 helix of the C-terminal domain of Tim44 confer resistance to MB-10.  MB-10 fits in a binding pocket in the C-terminal domain of Tim44.  MB-10 Inhibits Protein Import into Mammalian Mitochondria. MB-10 Inhibits Biogenesis of SOD2 in Mammalian Culture Cells.[1]

MB-10 Treatment Impairs Cardiac Development and Induces Apoptosis in Zebrafish Embryos [1]
Mutations in DNAJC19, a component of the PAM complex, lead to dilated cardiomyopathy with ataxia syndrome, an autosomal recessive Barth syndrome-like condition in patients. Researchers applied MB-10 to zebrafish embryos to determine how impaired TIMM44 altered development, particularly focusing on cardiac tissue. A transgenic zebrafish line in which DsRed is targeted to mitochondria under control of the heart specific cardiac myosin light chain promoter cmlc2 was used. Embryos were placed in either 1% DMSO, MB-10, MB-10.2, or analog 4 as indicated (Fig. 9) at 3 hpf and allowed to develop until 72 hpf. Embryos incubated with 10 μm MB-10 displayed dorsal body curvature and impaired heart development with excessive pericardiac effusion (Fig. 9) that is characteristic of zebrafish with dilated cardiomyopathy. MB-10-treated embryos also showed an increase in apoptotic cells as visualized by acridine orange staining (Fig. 9, highlighted by arrowheads), consistent with the increased apoptosis observed in MB-10-treated HeLa cells (Fig. 8, D–G). In addition, embryos incubated with MB-10.2 had developmental defects similar to MB-10 (Fig. 9C), whereas embryos incubated with analog 4 looked similar to the zebrafish treated with DMSO (Fig. 9D). Thus, MB-10 is an effective tool for characterizing TIMM44 function in cultured cells and zebrafish as well as yeast.

Yeast MIC50 assays. [1]
An early-log growth of the indicated yeast strains in YPEG media was diluted to an initial OD600 of 0.01 in fresh YPEG media. This diluted stock was dispensed in 50 μl aliquots into individual wells of a 96-well glass bottom plate. Subsequently, MB-10 in DMSO (or DMSO alone) in YPEG (50 μl) was serially diluted by a factor of 2 to the indicated concentration. The final concentration of DMSO was 1%. Plates were then incubated at 25˚C in a humidified chamber for 24 hours. During this time, the OD600 reached approximately 0.5. Each plate was shaken in a Beckman orbital shaker to resuspend settled cells, and the OD600 in each well was read by a Wallac Victor plate reader. For studies in Figure S1E, the OD600 of the WT strain in YPEG with 1% DMSO was set as 100% survival. For studies in Figure 4c, the OD600 of the WT strain in YPEG with 1% DMSO was set as 100% survival and all strains grew at approximately the same rate.

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Import of radiolabeled proteins into mitochondria and crosslinking assays. [1]
Prior to import into purified mitochondria, [35S]-methionine and cysteine labeled precursor proteins were generated with the TNT Quick Coupled Transcription/Translation kit. Import reactions with yeast mitochondria were conducted according to established methods. Because the compounds are typically soluble in DMSO, the final DMSO concentration of 1% was used, unless noted. The compounds in DMSO or DMSO vehicle were added to the mitochondria (25-50 µg/ml) in import buffer and incubated for 15 min incubation at 25°C. Import reactions were then initiated by the addition of precursor. Aliquots were removed at intervals during the reaction time-course and import was terminated with 25 g/ml trypsin on ice. After 15 min, 200 µg/ml soybean trypsin inhibitor was subsequently added. Mitochondria were pelleted by centrifugation at 8,000 x g for 5 min. Crosslinking assays were reported previously. Following import in the presence of 1 µM methotrexate (MTX), import-arrested precursor was subjected to crosslinking with 200 µM disuccinimidyl suberate (DSS) for 30 min on ice. After quenching the crosslinking reactions with 100 mM Tris, mitochondria were pelleted by centrifugation at 10,000 x g for 10 min and analyzed by SDS-PAGE and autoradiography.

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Oxygen consumption assays. [1]
Oxygen consumption of WT mitochondria was measured using a Clark-type oxygen electrode as described previously. Briefly, state II respiration was induced to a suspension of 100 µg/ml mitochondria in 0.23 M sucrose, 20 mM KCl, 20 mM Tris-HCl, 0.5 mM EDTA, 4 mM KH2PO4 and 3 mM MgCl2, pH 7.2 by adding 2 mM NADH. The consumption rate was monitored for approximately 2 min. Small molecule or DMSO was added to a final vehicle concentration of 1% and respiration was measured for another approximately 2 min. Uncoupled respiration was achieved by adding 20 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to the chamber.

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Target identification using the protease protection assay. [1]
40 µg of isolated yeast mitochondria were lysed with buffer containing 20 mM Hepes pH 7.4, 50 mM KCl, and 0.2 % Triton X-100, followed by centrifugation at 20,000 x g for 10 min. The lysate was treated with 1% DMSO or compound at 4°C for 15 min. and then incubated with 0.6 µg/ml of pronase at 25°C. One quarter of the reaction was removed at each time point, and pronase was immediately quenched by the addition of Laemmli sample buffer, followed by incubation at 95°C for 15 min. 1 µg of recombinant Tim44 was treated with 1 % DMSO or small molecule in buffer containing 20 mM Hepes-KOH pH 7.4, 50 mM KCl, and 0.01% BSA for 15 min at 4°C. The proteins were treated with pronase as indicated at 25°C for 15 min.

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Isolation of MB-10 resistant TIM44 mutants. [1]
The TIM44 gene including 300 bp 5’ and 3’ of the open reading frame was cloned into pRS314 for mutagenesis. TIM44 was mutagenized using error-prone PCR as described previously, the buffer contained Mg2+ and the concentration of the adenine nucleotide was decreased 5-fold. The mutagenized fragment was co-transformed with the linearized vector pRS315 into the Δtim44 strain that expressed TIM44 from the centromeric URA3 vector pRS316 (designated the shuffling strain) and transformants were selected on media lacking tryptophan. The transformants were subsequently transferred to plates with 5-fluoroorotic acid (FOA) to remove the pRS316-TIM44 plasmid. Cell harboring tim44 mutants were selected for their ability to grow in the presence of 15 µM MB-10. The mutant tim44 plasmids were subsequently recovered and sequenced and retransformed to confirm that resistance to MB-10 was plasmid dependent. tim44 mutants were reconstructed with single mutations MB-10 resistant mutations (T290S and I297V) and mutations of predicted MB-10 coordinating points(H292A, W316A, W417A, W316Y and W417Y) were synthesized.

Cell viability assay [1]
Cell Types: Integrate the plasmid expressing Su9-Ura3-myc into the LEU2 locus of WT and tim23-2 strains.
Tested Concentrations: 100μM.
Incubation Duration: 30 minutes.
Experimental Results: The WT strain expressing Su9-Ura3 failed to grow, while the tim23-2 mutant expressing Su9-Ura3 grew faster. The strains grew at similar rates when the medium was supplemented with uracil.

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Cell viability assay [1]
Cell Types: HeLa cells.
Tested Concentrations: 0-100 μM.
Incubation Duration: 24 hrs (hours).
Experimental Results: Inhibited HeLa cell viability, IC50 was 17.2 μM.

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Cell viability assays. [1]
Cell viability was measured with an MTT-based toxicology assay kit. HeLa cells were grown in 24-well dishes to 80% confluency. Cells were treated with 1% DMSO or various concentrations of MB-10 for 24 hr. After treatment, cells were incubated with MTT solution for additional 4 hr as described in the manufacturer protocols.

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Immunoprecipitation. [1]
20 g of isolated yeast mitochondria were lysed with buffer containing 20 mM Hepes pH 7.4, 80 mM KCl, and 0.2 % Triton X-100, followed by centrifugation at 20,000 x g for 10 min. The lysate was treated with 1% DMSO or 100 µM MB-10 at 25°C for 15 min. After small molecule treatment, 5 mM MgCl2 and 1 mM ATP were added to the lysate, followed by immunoprecipitation with anti-mHsp70 antibody and Protein A Sepharose beads. Tim44 was detected by immunoblot analysis.

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Pulldown assays. [1]
Mitochondria (400 μg) were pretreated with 1% DMSO or the indicated concentration of MB-10 for 15 min and subsequently lysed in buffer A [1% digitonin, 20 mM K+HEPES (pH 7.4), 80 mM KCl, 10% glycerol, and 1mM PMSF], followed by centrifugation at 20,000 x g for 30 minutes to remove insoluble material. 50 μg lysate was removed from the supernatant to use as a reference for the total sample (T). The remaining 350 μg was diluted to 0.4 μg/μl buffer A. Pulldowns were performed for 2 hr at 4˚C with rotation using 30 ul of HisPur Ni2+ beads. After binding, 50 μg of lysate was removed as a reference for the material that did not bind (denoted flow-through, FT). The beads were washed 4 times with buffer. The samples were eluted in Laemmli sample buffer supplemented with 15% β-mercaptoethanol and 300 mM imidazole. The eluate (E), total, and flow through were separated by SDS-PAGE and analyzed by immunoblotting.

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Cell manipulations. [1]
For microscopy experiments, HeLa cells treated with DMSO, MB-10 , or CCCP for 24 hrs were fixed with 4% formaldehyde and permeabilized with ice-cold methanol. Immunostaining was performed with rabbit anti-cytochrome c antibody and Alexa fluor 488 goat anti-rabbit IgG. Cells were visualized with a microscope 9Axiovert-200M using a Plan-Fluor 63x oil objective. Images were acquired at room temperature with a charge-coupled device camera controlled by Axiovision software.

Zebrafish manipulations. [1]
Zebrafish displaying fluorescent hearts were derived from transgenic TL fish expressing a fusion of the CoxIV targeting sequence with DsRed regulated by a cmlc2 (cardiac myocyte light chain-2) promoter. Lines were maintained in a 14-hr light/10-hr dark cycle and mated for one hour to obtain synchronized embryonic development. Embryos were grown for 3 hpf in E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM Mg2SO4) and then incubated with E3 buffer supplemented with 1% DMSO, MB-10, MB-10.2, or Analog-4 for 3 days at 28.5°C. Following treatment, embryos were imaged using a Leica MZ16F fluorescent stereoscope (TexasRed filter set) at 5X magnification. Alternatively, 3-day embryos were stained with 10 µg/ml acridine orange and incubated for 30 min. Embryos were then washed with E3 buffer to remove residual stain, and apoptotic cells were likewise imaged using a Leica MZ16F fluorescent stereoscope (FITC filter set).

[1]. Adaptation of a Genetic Screen Reveals an Inhibitor for Mitochondrial Protein Import Component Tim44. J Biol Chem. 2017 Mar 31;292(13):5429-5442.

We completed a screen to identify inhibitors of the general import pathway by adapting a genetic screen in yeas. Because this assay selects for a gain in growth, false-positive compounds that are generally lethal are omitted. Metrics for evaluating and validating a robust high throughput include a low hit rate typically less than 0.14% and a screening window coefficient (referred to as the Z′ factor) greater than 0.5. Our screening approach was strong with a high Z′ factor (>0.8) and low hit rate (0.02%). Another benefit of this screening approach is that a WT yeast strain, instead of mutants, was used. In addition, as shown with previous inhibitors MB-1 and MB-6, MB-10 inhibits import in both yeast and mammalian mitochondria. The SAR studies in the protein import assays (Fig. 3) and zebrafish (Fig. 9) support that MB-10 seems specific for targeting Tim44. Indeed, MB-10 and MB-10.2 specifically induced cardiac deficiency in developing zebrafish embryos and inhibited import. In contrast, analog 4 did not alter zebrafish development, although the compound shared a similar structure to MB-10 and MB-10.2 (Fig. 9). Thus, these probes are valuable for study in a broad range of model systems.[1]
A multifaceted strategy illustrates that MB-10 likely targets Tim44, but interactions at the interface with other components of the PAM and TIM23 complex cannot be ruled out completely. With a genetic approach, the tim23-2 mutant was more sensitive to MB-10 than the tim10-1 mutant. A battery of import assays using substrates of each translocon (TOM, TIM23, and MIA (mitochondrial intermembrane space assembly)) narrowed the candidate to five or six factors of the PAM complex. Subsequent detailed studies including cross-linking to the cyt b2(167)-DHFR and protease protection corroborated that MB-10 likely targeted Tim44. Whereas the direct approach of incorporating MB-10 into crystallography studies was not successful, a genetic approach identified Tim44 mutants that were not sensitive to MB-10. In conjunction with docking and MD simulations, MB-10 binds in a pocket in the C-terminal region that is on the opposite face from a large groove that may interact with the membrane. The N-terminal region of Tim44 interacts with motor partners Hsp70, Mge1, Pam18, and Pam16, but our study shows that the C-terminal region is also essential for Tim44 function. Indeed, the C-terminal region is highly conserved, mutations at invariable residues resulted in a dominant-negative phenotype, and MB-10 addition blocked interactions of Tim44 with the imported precursor and Hsp70. As shown in Fig. 7D, we propose that MB-10 blocks binding of the C-terminal domain of Tim44 and the precursor, thereby blocking protein translocation. The cross-linking studies indicate that binding of precursor to Hsp70 increases, but this intermediate is not productive for protein import. Based on our pulldown studies, MB-10 does not disrupt interactions among components of the TIM23 translocon and PAM complex. Our studies thus agree with those of Mokranjac and co-workers in which they reported that the C-terminal domain interacted with the translocating protein. Thus, MB-10 is a specific probe to block interactions of the translocating precursor with the PAM complex.[1]
From the import studies with substrates that have a stop transfer motif, MB-10 at 100 μm inhibited the import of precursors sorted to the intermembrane space (Fig. 3). This import inhibition is similar to the phenotype of tim44 temperature-sensitive mutants reported previously. Recently, the role of the import motor in the translocation of precursors that are laterally sorted (i.e. cyt b2(167)-DHFR and cyt c1) has been under debate. The “modular” model proposes that the TIM23 complex exists in two forms: one with Tim21 that lacks the import motor and mediates lateral insertion and the second one with the import motor that lacks Tim21 and mediates transport into the matrix. The “single-entity” model proposes that all essential subunits of the translocase function as one complex that may be actively remodeled based on targeting sequences of the translocating substrate. Given that MB-10 inhibits the import of cyt b2(167)-DHFR and cyt c1, our studies are tilted to support the “single-entity” model, because Tim44 of the motor is required for the import of laterally sorted precursors. MB-10 will be a useful tool to continue mechanistic studies about how the dynamic TIM23 translocon mediates protein import.[1]

C12H8FN3O3S

293.273624420166

293.03

C, 49.15; H, 2.75; F, 6.48; N, 14.33; O, 16.37; S, 10.93

394694-98-5

394694-98-5

6895129

Light yellow to yellow solid powder

3.1

1

6

3

20

402

0

C1=CC(=CC(=C1)F)C(=O)N/N=C/C2=CC=C(S2)[N+](=O)[O-]

QDYQZMWFIYLMNX-VGOFMYFVSA-N

InChI=1S/C12H8FN3O3S/c13-9-3-1-2-8(6-9)12(17)15-14-7-10-4-5-11(20-10)16(18)19/h1-7H,(H,15,17)/b14-7+

3-fluoro-N-[(E)-(5-nitrothiophen-2-yl)methylideneamino]benzamide

MitoBloCK 10; MitoBloCK-10; MB10; MitoBloCK-10; 3-Fluoro-N'-((5-nitrothiophen-2-yl)methylene)benzohydrazide; 394694-98-5; 3-fluoro-N'-[(E)-(5-nitrothiophen-2-yl)methylidene]benzohydrazide; 3-fluoro-N-[(E)-(5-nitrothiophen-2-yl)methylideneamino]benzamide; MB-10; MB 10; MB-10

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~125 mg/mL (~426.2 mM)

Solubility in Formulation 1: 2.08 mg/mL (7.09 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)