MeCP2 is a naturally supercharged protein with cell membrane transduction capabilities

Abstract

The intrinsically disordered protein MeCP2 is a global transcriptional regulator encoded by the MECP2 gene. Although the structured domains of MeCP2 have been the subject of multiple studies, its unstructured regions have not been that extensively characterized. In this work, we show that MeCP2 possesses properties akin to those of supercharged proteins. By utilizing its unstructured portions, MeCP2 can successfully transduce across cell membranes and localize to heterochromatic foci in the nuclei, displaying uptake levels a third lower than a MeCP2 construct fused to the cell-penetrating peptide TAT. MeCP2 uptake can further be enhanced by the addition of compounds that promote endosomal escape following cellular trafficking by means of macropinocytosis. Using a combination of in silico prediction algorithms and live-cell imaging experiments, we mapped the sequence in MeCP2 responsible for its cellular incorporation, which bears a striking resemblance to TAT itself. Transduced MeCP2 was shown to interact with HDAC3. These findings provide valuable insight into the properties of MeCP2 and may be beneficial for devising future protein-based treatment strategies.

Keywords: MeCP2; TAT fusion proteins; cell penetrating peptides; supercharged protein.

FIGURE 1

MeCP2 sequence alignment, constructs and SDS–PAGE. (a) Sequence alignment of full‐length MeCP2 (e2 isoform), minMeCP2, and tMeCP2. Sequences corresponding to the MBD and NID are colored in light and dark purple, respectively. Sequences that may be responsible for MeCP2 uptake are boxed, the nuclear localization sequence is underlined and marked with arrows. (b) Schematic representation of the MeCP2‐eGFP, TAT‐MeCP2‐eGFP, minMeCP2‐eGFP, MeCP2ΔM3‐eGFP, and MeCP2ΔM2‐eGFP constructs and their appropriate acronyms. (c) SDS–PAGE of MeCP2‐eGFP, TAT‐MeCP2‐eGFP, and minMeCP2‐eGFP, along with the peqGOLD V protein marker. (d) SDS–PAGE of MeCP2ΔM3‐eGFP and MeCP2ΔM2‐eGFP along with the peqGOLD IV protein marker.

FIGURE 2

Investigation of MeCP2 uptake. Live‐cell imaging of NIH3T3 cells incubated with 4 μM MG, TMG, minMG, TG, or MeCP2 buffer. Scale bar = 20 μm.

FIGURE 3

(a) Representative imaging flow cytometry images of NIH3T3 cells incubated with 3 μM MG, TMG, minMG. (b) Quantification of TMG, MG, and minMG (3 μM each) nuclear uptake using imaging flow cytometry. The data represents mean values ± SDs of three biological replicates. ANOVA was used to determine significant differences between protein variant uptake levels. ns, not significant; *p ≤ 0.05; **p ≤ 0.01.

FIGURE 4

Endosomal involvement in MG and TMG uptake. (a) Live‐cell imaging of NIH3T3 cells incubated with 4 μM MG (top) and 4 μM MG co‐incubated with 100 μM chloroquine (bottom). Scale bar = 20 μm. (b) Quantification of cellular uptake in NIH3T3 cells treated with 4 μM MG and 4 μM TMG and buffer‐treated cells (dark gray) alone and in combination with 100 μM chloroquine (+ chl) or 80 mM sucrose (+ suc). The data are presented as the means ± SDs of three biological replicates. Student's t test was used to determine the significance of the differences between MG and TMG uptake upon the addition of endosome‐disrupting compounds; *p ≤ 0.05, **p ≤ 0.01. (c) Quantification of uptake of 4 μM MG and 4 μM TMG alone or in combination with 0.5 mM amiloride into NIH3T3 cells. (d) Quantification of uptake of 4 μM MG and 4 μM TMG alone or in combination with 0.5 mM indomethacin into NIH3T3 cells. Student's t test was used to determine the significance of the differences between MG and TMG uptake upon the addition of endocytosis inhibitors; *p ≤ 0.05, **p ≤ 0.01.

FIGURE 5

Live‐cell imaging of NIH3T3 cells incubated with putative MeCP2 constructs lacking the M3 or the M2 sequence. (a) Live‐cell imaging of NIH3T3 cells incubated with 3 μM MΔM3G (top), MΔM2G (bottom). Scale bar = 20 μm. (b) Comparison of MG and MΔM3G uptake (3 μM each) using imaging flow cytometry. The data represents mean values ± SDs of three biological replicates. ANOVA was used to determine significant differences between protein variant uptake levels; ns, not significant; *p ≤ 0.05.

FIGURE 6

CoIP of MG and TMG. MG and TMG (top panels) both recruit HDAC3 (bottom panels) following transduction into intact cells (left panels, protein concentrations employed are 1.5 μM for each construct) as well as when added directly to the cell lysate (right panels, protein concentrations employed are 150 nM for each construct). M—Chameleon DUO pre‐stained marker.