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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 15, 2004 as doi:10.1096/fj.04-1982fje. |
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* Department of Pharmacology, Joan and Sanford I. Weill Medical College of Cornell University, New York, New York, USA; and
Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Montreal, Quebec, Canada
1Correspondence: Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021, USA. Email: hhszeto{at}med.cornell.edu
SPECIFIC AIMS
Fluorescent labels are commonly used to investigate the mechanisms of cellular uptake and intracellular distribution of cell-penetrating peptides. However, labels such as fluorescein and rhodamine are relatively large and very lipophilic, and may significantly alter physicochemical properties of small peptides. To minimize the impact of the fluorescent probe on a tetrapeptide, we substituted one of the amino acids (Lys4) in a tetrapeptide ([Dmt1]DALDA, Dmt-D-Arg-Phe-Lys-NH2 where Dmt=2',6'-dimethyltyrosine) with two different fluorescent amino acids (ß-dansyl-L-
,ß-diaminopropionic acid (dnsDap4) or ß-anthraniloyl-L-,ß-diaminopropionic acid (atnDap4). Initial studies with confocal laser scanning microscopy (CLSM) showed very different localization patterns for the two fluorescent analogs, with [Dmt1,atnDap4]DALDA showing mitochondrial localization and [Dmt1,dnsDap4]DALDA showing diffuse cytoplasmic localization. We then carried out additional studies with isolated mitochondrial preparations to determine the reasons behind their differences and which fluorescent analog better reflects the targeting and function of the parent peptide.
PRINCIPAL FINDINGS
1. CLSM showed different intracellular localization for [Dmt1,dnsDap4]DALDA and [Dmt1,atnDap4]DALDA
To examine uptake and localization of the two fluorescent analogs, Caco-2 cells were incubated with 0.1 µM of the fluorescent analogs for 15 min at 37°C and CLSM carried out with living cells. Rapid cellular uptake was observed within 15 min for [Dmt1,dnsDap4]DALDA and [Dmt1,atnDap4]DALDA. Neither fluorescent analog showed vesicular distribution and both were excluded from the nucleus. However, the two fluorescent analogs showed very different intracellular distribution, with [Dmt1,dnsDap4]DALDA distributed diffusely throughout the cytoplasm (Fig. 1
A), while the localization pattern of [Dmt1,atnDap4]DALDA showed a perinuclear localization pattern that overlaps that of Mitotracker TMRM (tetramethylrhodamine methyl ester), suggesting mitochondrial distribution (Fig. 1B
).
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2. [3H][Dmt1]DALDA and [Dmt1,atnDap4]DALDA are localized to inner mitochondrial membrane
The uptake of [Dmt1,atnDap4]DALDA by mitochondria was confirmed by fluorescence quenching studies using isolated mouse liver mitochondria. Addition of mitochondria to a solution of [Dmt1,atnDap4]DALDA resulted in immediate and sustained quenching of the fluorescent signal (Fig. 1C
). In contrast, although the fluorescent signal of [Dmt1,dnsDap4]DALDA was immediately quenched upon addition of mitochondria, this was rapidly reversed within seconds and full recovery of the fluorescent signal was observed within 60 s. Incubation of isolated mitochondria with [3H][Dmt1]DALDA also resulted in the accumulation of radiolabel in the mitochondrial pellet (46.5±0.3 pmol/mg protein in 8 min), and mitochondrial subfractionation studies revealed that 85% of the radiolabel was associated with the inner mitochondrial membrane (Fig. 1D
).
3. [Dmt1,dnsDap4]DALDA caused mitochondrial depolarization and swelling
We then determined whether the targeting of [Dmt1]DALDA and [Dmt1,atnDap4]DALDA to mitochondria might alter mitochondrial function. Mitochondrial potential can be monitored with the use of TMRM which is taken up by mitochondria in a potential-driven manner. Addition of 100 µM [Dmt1]DALDA or [Dmt1,atnDap4]DALDA had no effect on TMRM fluorescence, indicating that these two peptides did not alter mitochondrial potential (Fig. 2
A). Surprisingly, the addition of 100 µM [Dmt1,dnsDap4]DALDA led to mitochondrial depolarization (Fig. 2A
). Mitochondrial depolarization can lead to the opening of a nonspecific pore in the inner membrane (mitochondrial permeability transition) and mitochondrial swelling. Addition of 100 µM [Dmt1,dnsDap4]DALDA to a mitochondrial suspension caused rapid onset of mitochondrial swelling as measured by absorbance at 540 nm (A540), whereas addition of 100 µM [Dmt1]DALDA or [Dmt1,atnDap4]DALDA had no effect (Fig. 2B
).
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4. [Dmt1]DALDA and [Dmt1,atnDap4]DALDA protects against mitochondrial swelling induced by calcium overload
Calcium overload can cause mitochondrial permeability transition and swelling (Fig. 2C, D
). Pretreatment of mitochondria with 100 µM [Dmt1]DALDA or [Dmt1,atnDap4]DALDA before addition of Ca2+ resulted only in transient depolarization (Fig. 2C
) and prevented mitochondrial swelling (Fig. 2D
). In contrast, [Dmt1,dnsDap4]DALDA was unable to protect against Ca2+-induced depolarization or mitochondrial swelling.
CONCLUSIONS AND SIGNIFICANCE
The present findings were quite unexpected and clearly show that incorporation of even very small fluorescent labels can significantly alter intracellular targeting and function of small peptides. The studies with isolated mitochondria help explain the difference in intracellular localization of the two fluorescent analogs, and a proposed model is presented in Fig. 3
. Our results suggest that [Dmt1,dnsDap4]DALDA is taken up into the mitochondrial matrix where the accumulation of a cationic molecule would result in depolarization, swelling of the mitoplast and rupture of the outer mitochondrial membrane. This will lead to the efflux of [Dmt1,dnsDap4]DALDA from mitochondria, accounting for its diffuse cytoplasmic distribution in the whole cell studies. Thus [Dmt1,dnsDap4]DALDA is a mitotoxic molecule. In addition, mitochondrial swelling can lead to the release of cytochrome c from mitochondria and cytochrome c has been shown to cause activation of the caspase cascade leading to apoptosis.
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In contrast, [Dmt1,atnDap4]DALDA and [Dmt1]DALDA are associated with the inner mitochondrial membrane. Because they do not penetrate the mitochondrial matrix, they do not cause depolarization or mitochondrial swelling. Furthermore, both [Dmt1,atnDap4]DALDA and [Dmt1]DALDA protected mitochondria against Ca2+-induced permeability transition and swelling, making them mitoprotective agents.
These findings clearly demonstrate that the incorporation of a fluorescent label can dramatically alter the cellular uptake and intracellular targeting of small peptides, and profoundly alter their biological functions. These studies also highlight how confocal imaging data may be easily misinterpreted. Without the isolated mitochondria studies, and by using only [Dmt1,dnsDap4]DALDA, we might have simply concluded that [Dmt1]DALDA is transduced across the plasma membrane and distributed freely throughout the cytoplasm. Experimental results obtained with fluorescent labels needs to be interpreted with caution, and the use of multiple fluorophores, together with confirmation using the parent peptide or radiolabeled peptide, is recommended.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1982fje;
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