NPC1 plays a role in the trafficking of

specific cargo to melanosomes

Alina Adriana Rus, Ioana V. Militaru, Ioana Popa, Cristian V. A. Munteanu, Livia Elena Sima, Nick
Platt, Frances M. Platt, and Stefana M. Petrescu

Presented by - Janki Patel Published in - JBC

MSc - MBT (Previous) Published on - 7 July, 2023
 Why did I choose this paper?

• This paper provides the insists into trafficking of specific cargo to

melanosomes by NPC1 protein.
• I was interested to know how skin pigmentation process under
physiological conditions can be done and correlate it with the NPC1
disease.
 ABBREVIATIONS
• ACN - Acetonitrile
• ApoE - Apolipoprotein E
• DCT - Dopachrome-Tautomerase
• DOPA, 3,4 - Dihydroxy-L-Phenylalanine 3,4
• Endo H - Endoglycosidase H
• ER - Endoplasmic Reticulum
• GlcCer - Glucosylceramidase
• GSL - Glycosphingolipids
• HMB - Human Melanoma Black
• LAMP - Lysosome Associated Membrane Protein
• LRO - Lysosome-related Organelles
• MITF - Microphthalmia-Associated Transcription Factor
• NPC1 - Niemann–Pick disease type C1
• PNGase F - Peptide-N Glycosidase F
• TGN - Trans-Golgi Network
• TYR - Tyrosinase
• TYRP - Tyrosinase-Related Protein.
 PREVIOUS STUDIES
• Gong, X., Qian, H., Zhou, X., Wu, J., Wan, T., Cao, P., et al. (2016) Structural insights
into the niemann-Pick C1 (NPC1)-mediated cholesterol transfer and ebola infection. Cell
165, 1467–1478
• Evans, W. R. H., and Hendriksz, C. J. (2017) Niemann-Pick type C disease the tip of the
iceberg? a review of neuropsychiatric presentation, diagnosis and treatment. BJpsych
Bull. 41, 109–114
• Wheeler, S., and Sillence, D. J. (2020) Niemann–Pick type C disease: cellular pathology
and pharmacotherapy. J. Neurochem. 153, 674–692
• Höglinger, D., Burgoyne, T., Sanchez-Heras, E., Hartwig, P., Colaco, A., Newton, J., et al.
(2019) NPC1 regulates ER contacts with endocytic organelles to mediate cholesterol
egress. Nat. Commun. 10, 4276
• Chen, O. C. W., Colaco, A., Davis, L. C., Kiskin, F. N., Farhat, N. Y., Speak, A. O., et al.
(2020) Defective platelet function in Niemann-Pick disease type C1. JIMD Rep. 56, 46–
57
• Le, L., Sirés-Campos, J., Raposo, G., Delevoye, C., and Marks, M. S. (2021)
Melanosome biogenesis in the pigmentation of mammalian skin. Integr. Comp. Biol. 61,
1517–1545
 INTODUCTION

What is NPC1 ?
• The Niemann–Pick type C1 (NPC1) gene encodes a multipass membrane glycoprotein found in
the limiting membrane of late endosomes/lysosomes. Before reaching the endolysosomal
compartment, the protein is synthesized in the endoplasmic reticulum (ER) and transported to the
Golgi and from the trans-Golgi network (TGN) to late endosomes.
• Loss-of-function mutations in the NPC1 gene are responsible for the Niemann–Pick disease type
C1 (NPC) characterized by the accumulation of glycosphingolipids, sphingomyelin, sphingoid
bases, and cholesterol in late endosomes.
• Although NPC1 function has not been precisely elucidated, it has been proposed that it
coordinates cholesterol efflux from lysosome toward other cell compartments such as the ER or
plasma membrane.
• Indeed, loss of NPC1 results in natural killer cells and platelet functional defects that have been
associated with reduced acidic store Ca2+ levels affecting the LRO secretion.
Melanosome Biogenesis
• Melanosome biogenesis is initiated
in multivesicular endosomes, also
called stage I melanosomes, where
PMEL fibrils start to assemble.
• In stage II melanosomes, PMEL
fibrils give the melanosomes their
characteristic ellipsoidal shape and
striated appearance.
• Both stage I and stage II
melanosomes are unpigmented.
• Melanin starts to be produced in
stage III melanosomes, to which
melanin synthesizing enzymes, such
as Tyrosinase or TYRP1, are
transported.
• To accomplish the pigmentation
process, the melanin is deposited on
PMEL17 fibrils, and contributes to
the maturation of melanosomes, the
pigmented organelles of the skin,
hair, and eyes.
 Role of TYR
• TYR is central in the pathway, catalyzing the rate-limiting step of melanogenesis and
in its absence melanin is not synthesized, as seen in patients with oculocutaneous
albinism type IA.
• As such, a defective processing of TYR, mutations in TYR gene, or an altered
transport to melanosomes can lead to pigmentation defects.
• TYR is a glycoprotein whose folding and maturation are highly dependent on the N-
glycosylation process that starts within the ER.
• Misfolded TYR mutants are recognized by EDEM1 and removed from the ER by
ER-associated degradation, targeting for proteasomal degradation.
• Folded TYR molecules are exported from the ER to the Golgi, where they undergo
complex N-glycosylation processing.
• TYR is transported to melanosomes from the Golgi via an endocytic pathway .
 HYPOTHESIS

Whether the biogenesis of another specialized LRO, the melanosome,

required for the pigmentation process occurring in melanocytes, could
also be affected by NPC1 loss?
 OBJECTIVE 1

Cell model for studying the influence of NPC1 protein

deficiency on pigmentation process
 In order to study the impact of NPC1 deficiency on cell pigmentation, they generated a
highly pigmented melanoma cell line (MNT-1) deficient in the NPC1 protein. The NPC1-
KO cells were generated using the CRISPR/Cas9- mediated gene targeting system and
underwent single clone selection and immunoblotting analysis.

A. Selected four out of the 30 clones tested that lacked NPC1 expression for further analysis. In
contrast to the MNT-WT cells, the NPC1-KO clones were devoid of the NPC1 protein, as shown by
Western blotting analysis.
 To evaluate relative lysosomal volume by flow cytometry:

B. Stained the four positive cell clones with Lysotracker green. In the NPC1-KO cells the lysosomal
volume was increased in all four clones at least two-fold relative to MNT-WT cells.
C and D. Confocal fluorescence microscopy staining with antibody against lysosome-associated
membrane protein (LAMP-2), further indicates an expanded late endosomal–lysosomal compartment
in NPC1-depleted cells.
 Thus, the lack of NPC1 protein leads to unesterified cholesterol and sphingolipid accumulation
within the lysosomal compartment. Moreover, other studies demonstrated that the transport of
TYR and TYRP-1 to the melanosome was influenced by glycosphingolipid level. Therefore, the
dysregulation of lipid pathways can impact the pigmentation process.

E. This was supported by the upregulation of LAMP-2 expression in the NPC1-KO cell line assessed
by the Western blot analysis.
F. Moreover, the mature form of cathepsin D determined by Western blotting was also elevated in
NPC1-KO cell line consistent with unaltered lysosomal function.
 Therefore, performed biochemical analysis to quantify the different
lipids storage in NPC1 deficient melanoma cells:

A. Cholesterol measurement by Amplex Red Assay shows increase in the NPC1-KO cell line relative
to WT control.
B-E. HPLC analysis of glycosphingolipids (GSLs)-derived glycans revealed that there was increase
in GM2, GM3, GD3, GM1b, GD1b, and GT1b. Depending on the cell line, different GSLs were
found to be accumulating in the endolysosomal pathway.
F. Despite the imbalance of different lipids in NPC1-KO cell line, Glucosyl ceramidase was present
at similar levels in both cell lines.
 RESULT 1

The characterization of NPC1-KO versus MNT-WT cells confirmed that

the newly created melanoma cell line recapitulates the NPC cellular
phenotypes in terms of increased lysosomal volume and intracellular
lipids accumulation.
 OBJECTIVE 2

The pigmentation process of MNT-1 melanoma cells is

impaired in the absence of NPC1
They noticed that in the absence of the NPC1 protein, the pigmentation
of the MNT-1 melanoma cell line is damaged.

A. MNT-WT cells were dark brown, almost black, whereas the color of the NPC1-KO cells was light
brown.
 To evaluate the fate of TYR in NPC1-KO cells:

B. Western blot analysis showed significantly lower levels of TYR in the two clones of NPC1-KO
cells analysed than the MNT-WT cells.
C. Moreover, NPC1 silencing by RNAi in MNT-WT cells recapitulated the observed changes at the
protein level, defined by a significantly decreased level of TYR.

D. By expressing NPC1 in KO cell line, the effect of NPC1 on TYR protein levels was reversed,
even though the differences were not statistically significant.
Interestingly, among several melanoma cell lines with different pigmentation status, NPC1 protein is
significantly more abundant in MNT-1 cells, the highly pigmented cells in which TYR is well
expressed than the amelanotic cells (A375 and SKMEL28) that lack mature TYR.
E. Co-immunoprecipitation studies using NPC1 antibody revealed a possible direct interaction between NPC1
and TYR.
F. Evaluate the activity of TYR in gel: The decreased activity of TYR in the NPC1 deficient cells is seemingly
linked to its reduced protein expression level.
 To investigate whether there is a downregulation of TYR at
transcriptional level:

G. They assessed its gene expression by real time quantitative reverse transcription PCR and the amount of
TYR mRNA in NPC1-KO cells was not significantly changed compared with the one in MNT-WT cells.
H. In addition, they have probed microphthalmia-associated transcription factor (MITF), the main
regulator of the melanogenesis enzymes gene expression, which did not show significant alteration
in KO cells.
Indeed, MITF levels in the two cell lines were comparable, suggesting that the absence of NPC1
does not induce modifications during transcription of the pigmentation genes.
 These results suggest that cholesterol is not the primary cause of pigmentation
defect in NPC1-KO cells.

Moreover, due to the cholesterol influence on pigmentation and MITF signaling, they tested whether cholesterol
depletion would rescue the phenotype in NPC1-KO cell lines.
A. Cells depleted in NPC1 displayed a significantly lower lysosome volume, compatible with the partial rescue
of the NPC phenotype upon treatment with 2-hydroxypropyl-β-cyclodextrin, an entrapping cholesterol drug used
in NPC treatment.
B and C. However, either the expression of TYR and PMEL17 nor the pigmentation status of these cells were
modified.
I and J. Furthermore, Western blot analysis revealed decreased levels of two other melanosomal
enzymes involved in melanin biosynthesis, namely TYRP-1 and DCT.
In addition to its downregulation, DCT protein exhibited a different glycosylation pattern in NPC1-
KO cells, with an increase in the low molecular band than the high molecular band.
 RESULT 2

Therefore, in the absence of NPC1, the pigmentation process is reduced,

associated with a reduction in the expression of TYR that is not modified
during transcription or translation, but rather at the posttranslational level.
 OBJECTIVE 3

Proteomic analysis of NPC1-KO cells by nanoLC-

tandem mass spectrometry
To gain further insights into the function of the NPC1 protein in melanogenesis,
they analyzed the WT and KO cells by mass spectrometry (MS)-based proteomics.

A. They used a method in which the cell lysates were separated in a first dimension by SDS-PAGE, the
separated bands were digested to peptides and injected into the nanoLC coupled MS. Principal component
analysis reveals two main distinct clusters corresponding to MNT-WT and NPC1-KO cells, respectively,
indicating the variation in protein expression between samples.
B. This proteomic-based approach confirmed the absence of NPC1 protein in the KO cell line and
revealed that 57 proteins were significantly modified in NPC1 deficient cells, with 27 proteins
upregulated and 30 proteins downregulated.
C. Among the significantly upregulated proteins in the NPC1-KO cell line, they found the lysosomal protein
LAMP-2 that was previously reported to be elevated in NPC1 deficient cells . They also identified significantly
elevated levels of proteins implicated in macromolecule biosynthetic processes and in intracellular transport.
D. Additionally, six proteins related to lipid metabolism were increased in the NPC1-KO cell line.
E. Interestingly, ApoE protein is also involved in the melanogenesis process as a regulator of PMEL17
maturation. The PANTHER classification system was used to categorize the own regulated proteins related to
their protein class and molecular function.
F. Moreover, some significantly decreased proteins in NPC1-KO cells are involved in vesicular transport.
Several proteins related to cell pigmentation that may show an impairment of the melanogenesis pathway were
plotted.
 RESULT 3

Taken together these data indicate that in the absence of the NPC1
protein, there were not dramatic differences at the proteome level,
although the expression of some proteins was enhanced while some
others were reduced.
 OBJECTIVE 4

TYR processing and degradation is altered in NPC1-

KO melanoma cells
Thus, preventing acidification along endolysosomal compartment by bafilomycin
partially restored TYR maturation and stability in NPC1-KO melanoma cells.

A. Cells were untreated or treated with bafilomycin A (100 nM, 5 h) and analyzed by Western blotting for TYR
N-glycan processing after Endo H and PNGase F glycosidase treatment of cell lysates.
B. Visual comparison of cell pellets of MNT-WT and NPC1-KO cells untreated or treated with bafilomycin A1.
 To gain insight into the TYR fate in the two cell lines, further evaluated the TYR
pool secreted by melanoma cells:

C. The culture media were collected after 72h and the extracellular vesicles were analyzed by Western blotting.
Although LAMP-2 is more secreted in NPC1-KO cells, TYR levels are lower in the secreted extracellular
vesicles. This indicates that the low expression level of TYR is not due to a higher pool in the extracellular
medium, but to an intracellular separation and degradation process.
Flotillin-1, which is an exosome component, was present in exosomes isolated from both melanoma cells,
suggesting that the secretion process is not impaired in the absence of NPC1.
 To further understand the TYR degradation process, they studied the response of
TYR expression levels to proteasome and lysosome inhibitors in NPC1-KO and
MNT-WT cells:

D. Cells were incubated for 5h with either MG132, a proteasomal inhibitor, or with a mix of pepstatin A and
leupeptin, to inhibit lysosomal enzymes.
In both treatment conditions, elevated levels of TYR were detected by Western blot, suggesting a proteasomal
but also a lysosomal degradation pathway of TYR in the two melanoma cell lines.
 Furthermore, the kinetics of TYR degradation in the two cell lines was assessed by
treatment with cycloheximide, a protein biosynthesis inhibitor that prevents
translational elongation:

E. MNT-WT and NPC1-KO cells were harvested at different time points (0, 0.5, 1, 2, 3, and 4 h) after
incubation with cycloheximide and the expression of TYR was examined by quantitative Western blot analysis.
The immunoblot and the quantification revealed a decreased half-life of TYR in the NPC1-KO cells (1h)
compared to MNT-WT (4h).
 RESULT 4

Therefore a late endosomes-TGN impaired traffic in the absence of NPC1

leading to a misrouting of TYR toward degradation.
 OBJECTIVE 5

TYR and PMEL17 fibrils are redistributed in immature

melanosomes in NPC1-KO cells
Co-localization of TYR with RAB38, TYRP-1, CD63, and PMEL17 (HMB-45)
by confocal immunofluorescence microscopy
 The abnormal localization of PMEL17, prompted to investigate whether there are differences in its
maturation process and fibril formation:

A. Found that the expression of the immature (P1/P2) and M-beta forms of PMEL17, recognized by PEP-13 antibody were not
modified in the absence of NPC1 protein. However, a ladder of fibril-associated fragments containing the repeat domain
(RPT)appeared highly expressed in the absence of NPC1. The residual insoluble PMEL17 aggregates, extracted in more
stringent detergent conditions, run with similar fibril patterns in gel. The immunoblot analysis showed that there was a
statistically significant increase in both soluble and insoluble PMEL17 forms in NPC1-KO compared with MNT1-WT cells.
B. The surface expression of PMEL17 protein measured by flow cytometry was similar in both cell
lines. Therefore, NPC1-KO cells display a raise in the mature PMEL17 protein, which is
accumulated in pre-melanosomes.

C. Of interest, Apolipoprotein E, a regulator of amyloid fibril generation had increased expression in

NPC1-KO cells in Western blot analysis consistent with the proteomics data.
Subcellular fractionation of MNT-WT and NPC1 cell homogenates on a sucrose gradient:

D. In which 12 fractions were collected. TYR in MNT-WT distributed in low- and high-density fractions over the gradient. One peak
corresponds to fractions in both cells, namely LAMP2 and NPC1 (fraction 3&4). A minor pool of TYR was also detected in the first fractions
abundant in EEA1 (fraction 1&2). In NPC1-KO cells the pool of TYR from the denser fractions were significantly depleted. On the other hand,
TYR peak associated with the low-density fractions enriched in premelanosomes was consolidated, in parallel with an increase in LAMP-2
content in these fractions. A similar profile was found for TYRP-1. The mature form of PMEL17, recognized by HMB-45 antibody displayed
an increased level but also an altered distribution in NPC1-KO cells.
 RESULT 5

The results suggest that in NPC1 deficient cells TYR and PMEL17 fibrils are found
in early stages (I–II) melanosomes and in other low-density compartments and that
the normal maturation of melanosomes is compromised.
SUMMARY
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