Department of Neurogenetics, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
Cyprus School of Molecular Medicine, Nicosia, Cyprus
Department of Neurogenetics
The Cyprus Institute of Neurology and Genetics
6 International Airport Avenue
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LRSAM1 Depletion Affects Neuroblastoma SH-SY5Y Cell Growth
and Morphology: The LRSAM1 c.2047-1G>A Loss-of-Function
Variant Fails to Rescue The Phenotype.
Deleterious variants in LRSAM1, a RING finger ubiquitin ligase which is also known as TSG101-associated ligase
(TAL), have recently been associated with Charcot-Marie-Tooth disease type 2P (CMT2P). The mechanism by which mutant
LRSAM1 contributes to the development of neuropathy is currently unclear. The aim of this study was to induce LRSAM1
deficiency in a neuronal cell model, observe its effect on cell growth and morphology and attempt to rescue the phenotype with
ancestral and mutant LRSAM1 transfections.
Materials and Methods
In this experimental study, we investigated the effect of LRSAM1 downregulation on
neuroblastoma SH-SY5Y cells by siRNA technology where cells were transfected with siRNA against LRSAM1.
The effects on the expression levels of TSG101, the only currently known LRSAM1 interacting molecule, were also
examined. An equal dosage of ancestral or mutant LRSAM1 construct was transfected in LRSAM1-downregulated cells
to investigate its effect on the phenotype of the cells and whether cell proliferation and morphology could be rescued.
A significant reduction in TSG101 levels was observed with the downregulation of LRSAM1. In addition,
LRSAM1 knockdown significantly decreased the growth rate of SH-SY5Y cells which is caused by a decrease in cell
proliferation. An effect on cell morphology was also observed. Furthermore, we overexpressed the ancestral and the
c.2047-1G>A mutant LRSAM1 in knocked down cells. Ancestral LRSAM1 recovered cell proliferation and partly the
morphology, however, the c.2047-1G>A mutant did not recover cell proliferation and further aggravated the observed
changes in cell morphology.
Our findings suggest that depletion of LRSAM1 affects neuroblastoma cells growth and morphology and
that overexpression of the c.2047-1G>A mutant form, unlike the ancestral LRSAM1, fails to rescue the phenotype.
Leucine Rich Repeat And Sterile Alpha Motif Containing
1 (Fig LRSAM1,), a RING finger E3 ubiquitin ligase,
participates in a range of cellular functions including
cell adhesion, signaling pathways and cargo sorting
through receptor endocytosis (1, 2), and is expressed in
both fetal and adult nervous systems (3). LRSAM1, also
known as TSG101-associated ligase (TAL), regulates the
metabolism of Tumor Susceptibility gene 101 (TSG101) by
attaching several monomeric ubiquitins to the C-terminus
of TSG101 (4). TSG101 is a tumor suppressor gene and
a component of the endosomal sorting complex required
for transport (ESCRT) machinery, with a significant role
in cell cycle regulation and differentiation, and is the only
reported interactor of LRSAM1 (2, 5).
Ubiquitination is an important process of the cellular
system, primarily regulating protein degradation by
proteasomes, endocytosis, transcription regulation, protein
trafficking and cell death (6, 7). Necessary components of
ubiquitination are three highly specific enzymes, namely
ubiquitin-activating enzyme (E1), ubiquitin-conjugating
enzyme (E2) and ubiquitin protein ligase (E3) (8). E2
enzymes form a complex with ubiquitin (E2-Ub) that is
pre-activated by E1 enzymes. The formed E2-Ub complex
is recognized and coupled by E3 ligases for ubiquitin
transfer to the target site (9). It has become evident that
E3 ubiquitin ligases play an essential role in the regulation
of axons, dendrites and dendritic spine morphogenesis as
well as in neuronal function (7, 10). Aberrant E3 ubiquitin
ligase activity has been associated with neurological
disorders, including mutations in PARK2 (Parkinson
Protein 2) and LRSAM1 which result in the juvenile type
of Parkinson’s disease (10) and the Charcot-Marie-Tooth
disease respectively (4).
LRSAM1 has recently been implicated in Charcot-
Marie-Tooth (CMT) pathways but its role remains unclear.
LRSAM1 knockdown in zebrafish has been reported to
cause delayed neurodevelopment (3), while LRSAM1
knockout mice presented only sensitization to acrylamideinduced
neurodegeneration with no anatomical or functional
abnormalities (11). Transfection of the c.2080C>T LRSAM1
variant in LRSAM1 knockout NSC34 mouse neuronal cells
caused axonal degeneration and also disrupted interaction of
LRSAM1 with RNA-binding proteins (12).
We reported a dominant LRSAM1 mutation (c.20471G>
A, p.Ala683ProfsX3) in a large Sardinian CMT type
2P family (13). To date, five other LRSAM1 mutations
have been associated with CMT neuropathy of which
one and four show recessive (4) and dominant (3, 12, 14,
15) inheritance respectively. All the dominant mutations
identified are located within the RING finger domain
whereas the recessive mutation is located 37 amino
acids upstream of this domain. To functionally examine
the effect of the dominant variant we had identified, we
downregulated LRSAM1 in neuroblastoma SH-SY5Y
cells and overexpressed the ancestral and the c.20471G>
A mutant LRSAM1 in LRSAM1 knocked down cells.
We hereby report the effects of LRSAM1 downregulation
on SH-SY5Y cells and the inability of mutant LRSAM1
to reverse the phenotype, contrary to the ancestral form.
Materials and Methods
In this experimental study, we investigated the effect of
LRSAM1 downregulation on neuroblastoma SH-SY5Y
cells followed by the overexpression of ancestral and
mutant LRSAM1 in these knocked down cells.
Human SH-SY5Y neuroblastoma cells culture
Human SH-SY5Yneuroblastoma cells (ECACC, Sigma-
Aldrich, USA) were cultured in DMEM (Invitrogen,
USA) without L-glutamine. The growth medium was
supplemented with 10% fetal bovine serum (FBS,
Invitrogen, USA), 2% GlutaMAXTM (Gibco, USA) and
1% Penicillin-Streptomycin 100X Solution (Invitrogen,
USA). SH-SY5Y cell lines were incubated at 37°C under
Whole human LRSAM1 constructs
The pIRES2-EGFP-LRSAM1 ancestral and mutant
constructs were purchased from Eurofins (Germany). Vectors
(pIRES2-EGFP) contained the whole coding and part of the
5´ and 3´ UTR of the human LRSAM1 cDNA in frame. NheI
and EcoRI restriction sites were added at the two ends. The
mutant LRSAM1 cDNA included a G base deletion at the
first codon of exon 25, representing the frameshift effect of
the c.2047-1G>A splice defect variant (13).
Cells were transfected using Lipofectamine® 3000
(Life Technologies, USA) according to the manufacturer’s
instructions. Lipofectamine® 3000 along with either
constructs or siRNA were dissolved in the Opti-MEM®
(Life Technologies, USA) reduced serum medium without
FBS and antibiotics.
Downregulation of LRSAM1 in neuroblastoma SH-SY5Y
For downregulation of LRSAM1, cells were doubletransfected
with siRNA (Table 1,) to ensure a more
efficient reduction of gene expression. The siRNA against
LRSAM1 (Life Technologies, USA) was transfected
into SH-SY5Y cells according to the manufacturer’s
instructions. Negative control siRNA (Life Technologies,
USA), lipofectamine only and untransfected-A were
used as controls of the experiments. Untransfected-A
represented cells that had not been transfected with siRNA
while the other two controls were used to detect any effect
of transfection. The cell growth rate was observed every
24 hours (Table 1,). Protein extraction was undertaken on
days 2 and 5.
Transfection of LRSAM1 constructs in knocked down
Knocked down SH-SY5Y cells were transfected on day
5 (Table 1,), using an equal, maximum recommended,
dose of ancestral or mutant LRSAM1 construct. The
empty vector (pIRES2-EGFP) was used as a control to
test for any possible effect of the construct on the cells.
Untransfected-B cells were also used as controls which
were treated with LRSAM1 siRNA but not transfected with
either ancestral or mutant LRSAM1 construct. Cells were
monitored using the IX73 inverted microscope (Olympus,
Japan) before and 48 hours after transfection.
Day 3-48 hours after 1st-24 hours after 2nd transfection
Day 4-72 hours after 1st-48 hours after 2nd transfection
Day 5-96 hours after 1st-72 hours after 2nd transfection
Cell concentration in knocked down SH-SY5Y cells
Cells were collected from each well using the appropriate
volume of 0.25% Trypsin-EDTA (Life Technologies,
USA). Concentration of live cells (number of cells/ml) was
determined using a hemocytometer (Hausser Scientific,
USA). Trypan blue 0.4% (Sigma-Aldrich, USA) was added
to the cell suspension (1:1 ratio) and only circular cells that
did not absorb the blue dye were counted.
Protein extraction from SH-SY5Y cells
Proteins were extracted using a protein lysis buffer
containing 1M NaCl, 10 mM Tris-Cl (pH=7.5), 10%
glycerol (Sigma-Aldrich, USA), 1% TweenTM20(Affymetrix,
USA), 10 Mm ß-mercaptoethanol (Sigma-Aldrich, USA)
and 1x EDTA-free Protease Inhibitor Cocktail (Promega,
USA). Lysates were sonicated, denatured at 95°C and
diluted in 1x sodium dodecyl sulfate 20 % (Fisher
Scientific, UK). The Coomassie Plus (Bradford) protein
assay (Thermo Scientific, USA) was used to measure the
Western blot analysis
Proteins were separated on SDS-PAGE (8-12%) gels and
transferred to PVDF membranes (Millipore, Germany).
Membranes were blocked with 5% non-fat milk in PBS0.1%
TweenTM20 and were incubated overnight at 4°C
with the respective specific primary antibody for each
protein [mouse anti-LRSAM1/Abcam ab73113 (1:400),
rabbit anti-LRSAM1/Novus Biological H00090678-D01
(1:750), rabbit anti-CASPASE-3/Santa Cruz SC7148
(1:700), mouse anti-CYCLIN D1/Abcam ab6152 (1:300),
mouse anti-TSG101/Novus Biological NB200-11 (1:500)
and mouse anti-ß-ACTIN/Sigma-Aldrich A2228 (1:4000)]
which was diluted in phosphate buffered saline (PBS)-0.1%
TweenTM20. The membranes were then incubated for 2 hours
with the appropriate secondary antibodies [AP124P goat
anti-mouse IgG-Peroxidase H+L/Millipore (1:7000) and
sc-2077 donkey anti-rabbit IgG-HRP/ Santa Cruz (1:7000)]
followed by incubation with the visualization LumiSensorTM
Chemiluminescent HRP Substrate Kit (Genscript, USA).
Membranes were finally visualized using the UVP imaging
system (BioRad, USA). Western blots were quantified using
the ImageJ software (https://imagej.nih.gov). Protein quantity
ratio was estimated relative to ß-actin.
Quantitative data (ratio %) from three independent
experiments were analyzed using the two-tailed Student’s
paired t test. A P<0.05 was considered statistically
significant. All the data are expressed as mean ± SD of
the three replicates. The mean of quantitative data of the
control samples was set to 100%.
This study was ethically approved by the Cyprus
National Bioethics Committee (ΕΕΒΚ/ΕΠ/2013/28).
Downregulation of LRSAM1 affects growth and
morphology of undifferentiated SH-SY5Y cells
The efficiency of downregulation was evaluated with
Western blot analysis on days 2 and 5. Twenty-four hours
after the first LRSAM1 siRNA transfection (day 2), we
observed a 41 ± 2.94% reduction of endogenous LRSAM1.
Downregulation of LRSAM1 also caused a 30 ± 4.99%
reduction in TSG101 levels (data not shown). Following
the double-transfection of LRSAM1 siRNA, a 75 ± 3.94%
and 47 ± 2.72% decrease in LRSAM1 and TSG101 levels
was observed (Fig .1,). Growth of LRSAM1 knocked down
cells was remarkably reduced 72 hours after the second
transfection (day 5). Slightly reduced growth was observed
in negative siRNA and lipofectamine only controls, reflecting
the transfection effect (Fig .2A,). However, downregulation
of LRSAM1 substantially decreased the growth rate of
neuroblastoma SH-SY5Y cells when compared with the
Monitoring the cells 72 hours after the first transfection,
we observed that LRSAM1 downregulation had affected the
morphology of the cells compared with negative control cells
(Fig .2B,). The majority of knocked down cells had a spherical
shape with only a small proportion displaying an elongated
shape. No neurite formation among the cells was observed in
knocked down cells. In addition to a higher density, negative
control cells not only showed an elongated shape, but they
also displayed short neurite outgrowths in empty spaces
among the cells with a tendency to form networks.
To identify the cause of the observed reduction in
growth rate, we examined the protein levels of two
protein markers with Western blot analysis. Cyclin D1
and Caspase-3 were used as the cell cycle and apoptotic
markers respectively. Equal expression of Caspase-3 was
detected in knocked down and control cells (Fig .3A,).
However, a significant reduction of 44 ± 4.50% of Cyclin
D1 expression was observed in knocked down cells
when compared with controls (Fig .3B,), thus indicating a
potential role for LRSAM1 in the cell cycle process.
Transfection of LRSAM1 in knocked down SH-SY5Y cells
Knocked down cells were transfected with ancestral or
mutant LRSAM1 constructs. Efficiency of the transfection
was monitored with Western blot analysis (Fig .4,). Given
that the efficiency of the knockdown was approximately
70%, 30% of the endogenous LRSAM1 protein level
was expected to be present. This level of endogenous
LRSAM1 was confirmed in the empty vector pIRES2EGFP
and the untransfected-B controls as well as those
transfected with the mutant LRSAM1. In the latter,
two bands were observed representing the ancestral
(endogenous) and the lower molecular weight truncated
mutant protein (exogenous). Total expression levels of
LRSAM1 (ancestral and mutant) in ancestral and mutant
transfected knocked down cells was equivalent (89 and
- siRNA-mediated downregulation of LRSAM1. A. Western blot analysis of endogenous LRSAM1 and TSG101 levels, compared with thecontrols (negative control siRNA, lipofectamine only and untransfected-A cells). LRSAM1 and TSG101 levels were determined 72 hours after thesecond transfection. ß-ACTIN was used as an internal control and B. Quantification of LRSAM1 and TSG101 was performed relative to ß-ACTIN.
Untransfected-A cell values (LRSAM1 and TSG101) were set to 100% to reflect the normal cell growth within the 5 days of the experimentalprocedure. LRSAM1 levels (purple colour): LRSAM1 siRNA transfected cells (25 ± 3.94%, P<0.004), negative control siRNA transfected cells (90 ±
2.89%, P<0.009), lipofectamine only transfected cells (94 ± 2.15%, P<0.008) and untransfected-A cells (100 ± 1.36%). TSG101 levels (light purplecolour): LRSAM1 siRNA transfected cells (53 ± 2.72%, P<0.007), negative control siRNA transfected cells (84 ± 2.20%, P<0.027), lipofectamine onlytransfected cells (93 ± 2.49%, P<0.040) and untransfected-A cells (100 ± 4.51%).
- Effect of LRSAM1 knockdown on SH-SY5Y cells. A. Double transfection of LRSAM1 siRNA was performed on days one and two of the experimental
procedure. Cell counts after every 24 hours from day zero (plating) are depicted. Negative control siRNA, lipofectamine only and untransfected-A cell
cultures were performed and used as control for the experiment and B. Microscopy analysis 72 hours after the first LRSAM1 downregulation in double
transfected LRSAM1 siRNA and negative control siRNA SH-SY5Y cells. Arrows heads show the formation of early neurites (scale bar: 200 µm).
- Investigation of apoptotic (caspase-3) and cell cycle (cyclin D1) markers in LRSAM1 knockdown SH-SY5Y cells. Western blot analysis of A. Caspase-3and B. Cyclin D1 levels compared with the controls (negative control siRNA, lipofectamine only and untransfected-A cells). Protein levels were determined
96 hours after the first transfection. ß-ACTIN was used as an internal control. Quantification of caspase-3 and cyclin D1 was performed relative to ß-ACTIN.
Untransfected-A cell values (caspase-3 and cyclin D1) were set to 100%. Caspase-3 levels: LRSAM1 siRNA transfected cells (96 ± 5.48%, P<0.018), negative
control siRNA transfected cells (100 ± 4.55%, P<0.02), lipofectamine only transfected cells (96 ± 2.58%, P<0.019) and untransfected-A cells (102 ± 5.31%).
Cyclin D1 levels: LRSAM1 siRNA transfected cells (44 ± 4.50%, P<0.005), negative control siRNA transfected cells (95 ± 3.99%, P<0.009), lipofectamine only
transfected cells (104 ± 3.33%, P<0.03) and untransfected-A cells (99 ± 2.92%).
- Evaluation of endogenous and exogenous LRSAM1 levels. A. Western blot analysis of LRSAM1 levels 48 hours after the LRSAM1 transfection in
knocked down SH-SY5Y cells. An approximately 30% of endogenous protein was detected. ß-ACTIN was used as an internal control and B. Quantification
of ancestral and mutant LRSAM1 was performed relative to ß-ACTIN. Untransfected-B cell values (LRSAM1) were set to 30% (endogenous LRSAM1
background). Ancestral LRSAM1 transfected cells representing ancestral-endogenous + ancestral-exogenous LRSAM1 levels within a single band (89 ±
1.80%, P<0.0087 relative to untransfected-B). LRSAM1 mutant transfected cells representing ancestral-endogenous (31 ± 0.68%, P<0.01) and mutant-
exogenous (59 ± 1.68%, P<0.007) LRSAM1 levels in two different size bands. Empty vector pIRES2-EGFP transfected cells representing ancestral-
endogenous levels (28 ± 0.71%, P<0.0045). Untransfected-B cells representing ancestral-endogenous LRSAM1 levels (30 ± 0.72%, P<0.0019).
- Investigation of cell morphology and proliferation after transfection of ancestral or mutant LRSAM1 in knocked down SH-SY5Y cells. Microscopy
analysis before and 48 hours after the transfection of ancestral or mutant LRSAM1 constructs. At the time of LRSAM1 transfection, cells expressed 30% of
the endogenous LRSAM1 level. Arrows show the formation of early neurites (scale bar: 100 µm).
We overexpressed ancestral and mutant LRSAM1
in knocked down cells to investigate whether cell
proliferation and morphology could be rescued. Forty-
eight hours after transfection of the ancestral LRSAM1,
the growth of cells was considerably recovered
when compared with controls. Cells transfected with
ancestral LRSAM1 re-proliferated, morphology of the
cells was partly retrieved and cells displayed a more
elongated shape. Also, cells initiated formation of early
neurites. Transfection with an equal dose of the mutant
LRSAM1 not only did not improve cell proliferation,
but also worsened the morphological features of cells
when compared with controls. Specifically, mutant
LRSAM1 transfection induced the formation of cell
clusters comprising small circular and thin elongated
cells. Empty vector pIRES2-EGFP and untransfected-B
cells displayed the same morphology and growth
prior to the transfection without any improvement in
density, neurite formation and alteration in the shape
of the cells (Fig .5,).
A relatively small number of variants in LRSAM1 have
been associated with CMT2P, however, the majority of
them are predicted to interfere with the RING domain
and in turn the E3 ubiquitin ligase activity of the protein.
Ubiquitination plays a central role within cells and its
disruption is expected to have an impact on the overall
wellbeing of the cell. We thus investigated the effect of
LRSAM1 depletion on cell growth and morphology using
neuroblastoma cells, a good in vitro model to study the
function of a gene that is associated with a neurological
condition, namely CMT2P neuropathy.
LRSAM1 incomplete (70%) knockdown severely
affected the growth and morphology of neuroblastoma
cells. However, failure to observe a phenotype in mouse
NSC34 cells despite complete knockout of LRSAM1
with the CRISPR/Cas9 system has been reported (11,
12). The mouse model which was homozygous for a
loss-of-function variant in Lrsam1 had no anatomically
or functionally detectable abnormalities with only a
sensitivity of peripheral motor axons to acrylamideinduced
degeneration (11). Unlike the mouse model, the
morpholino induced Lrsam1 zebrafish model had both
anatomically and functionally detectable abnormalities
(3). Thus, species- and/or cell/model-specific LRSAM1
regulation may explain the differences between the
effects of LRSAM1 depletion that we observed in human
neuroblastoma SH-SY5Y cells as opposed to the reported
effect in mouse NSC34 cells.
Given that LRSAM1 downregulation affected the
cell cycle process by observing a lower level of cyclin
D1, the G1-phase is most likely impaired. We also
observed a significant reduction of TSG101 levels with
downregulation of LRSAM1. In another study on TSG101
deficient cells, G1 and G1/S-phase cyclins D1 and E were
not affected, however the S and M phase cyclins A2 and
B1 were prominently decreased (16). We therefore suggest
that knockdown of LRSAM1 affects cell cycle regulation
in the G1-phase at an earlier stage than TSG101. The
two molecules may act directly or indirectly and through
interaction with each other or independently to cause
changes in the cell cycle.
LRSAM1, through its E3 ligase activity, regulates
the level of TSG101 by targeting it for ubiquitination
and degradation. Silencing of TSG101 has been shown
to decrease proliferation and cell growth of adult and
embryonic tissues (17). Early embryonic lethality was
also observed in homozygous knockout TSG101 mice
(18). Interestingly, strong overexpression of TSG101 also
prevented cell division and induced cell cycle arrest (19).
It therefore seems that TSG101 is tightly controlled and is
necessary for normal cell function with LRSAM1 having
a significant role in adjusting the TSG101 level.
Other E3 ubiquitin ligases have also been reported to be
involved in cell cycle regulation and neurodegeneration.
Two E3 ubiquitin ligase complexes are involved in cell
cycle regulation; SCF (SKP1/CUL1/F-box) and APC/C
(anaphase prompting complex) complexes mediate
ubiquitination and activation of cell cycle marker proteins
(20). In addition, deficiency in the E3 ubiquitin ligase
activity of TRIM2 has been reported to cause severe early-
onset axonal CMT2R (21). TRIM2 ubiquitinates the
neurofilament light chain and patient sural nerve biopsy
as well as TRIM2-gene trap mice studies have shown the
accumulation of neurofilaments inside the axons (21, 22).
Deregulation in the ubiquitin proteasome system (UPS),
consisting of E3 ubiquitin ligases, has also been associated
with neurodegenerative disorders. Parkin, an E3 ligase,
exerts a direct, confirmed impact on neurodegeneration
which leads to Parkinson disease (23). Decreased levels
of HRD1and Fbxo2 E3 ligases have also been reported in
tissues obtained from Alzheimer’s patients (24).
Other CMT genes, which lack E3 ubiquitin ligase
activity, have also been implicated in cell cycle
progression. The ancestral GDAP1 (CMT2K and
CMT4A) has been reported to rescue cell cycle delay
caused by Fis1 deficiency in contrast to mutant GDAP1
(25), and an extended cell cycle was observed in zebrafish
mutants with impaired PRPS1 expression, a gene that has
been associated with X- linked CMT (26).
In the second part of this study, we overexpressed
the ancestral and the c.2047-1G>A mutant LRSAM1 in
knocked down cells. Given that transfection with the
mutant form failed to rescue the phenotype and also
deteriorated the morphology of the cells, we speculate
that the c.2047-1G>A mutation, which leads to protein
truncation, dramatically affects the activity of the enzyme
rendering it unable to cause ubiquitination. A recent study
demonstrated that recombinant mutant LRSAM1 RING
proteins, carrying frameshift or missense mutations,
abolished the ligase activity of LRSAM1. Mutant proteins
failed to interact and form poly-ubiquitin chains with E2
enzymes, which is a crucial process for efficient target
ubiquitination (27). Transfection of LRSAM1 with a
missense variant in the RING domain also caused axonal
degeneration in LRSAM1 knockout NSC34 cells and
disrupted interaction of LRSAM1 with RNA-binding
Five of the six LRSAM1 mutations that have been
associated with CMT2P exert a dominant effect and are
all located within the RING domain, suggesting that the
E3 ligase activity of the protein is affected. The recessive
mutations are located upstream of the RING domain and
result in no detectable protein. A dominant-negative effect
of the dominant mutations is possible, with the presence
of the mutant protein interfering with the function of the
ancestral LRSAM1, given the similar clinical phenotype
between patients with dominant and recessive forms. This
possibility is supported by the findings of Hakonen et al.
(27), who demonstrated that LRSAM1 RING mutants
maintained the capacity to form heterodimers with the
ancestral LRSAM1 proteins. Another possibility of a
dominant-negative effect of mutant LRSAM1 may be
through its interaction with TSG101. LRSAM1 interacts
with TSG101 for proper ubiquitination of TSG101 (2)
and the interacting amino acids precede the RING domain
of the protein, and both mutant and ancestral LRSAM1
may therefore compete for TSG101 binding. The
interaction and formation of a complex between mutant
LRSAM1 and TSG101 is not affected by the disruption
of the LRSAM1 RING domain (27). In the absence of
ubiquitination efficiency caused by RING domain-mutant
LRSAM1, the mutant LRSAM1-TSG101 complex may
lead to blockage of the TSG101 ubiquitination pathway,
consistent with an alternative dominant-negative effect of
We suggest that depletion of LRSAM1 affects
neuroblastoma cells growth and morphology and that
overexpression of the c.2047-1G>A mutant, in contrast
to the ancestral LRSAM1, fails to rescue the phenotype.
This study has been financially supported by a grant of
the Cyprus TELETHON (Grant number: 73115). There is
no conflict of interest in this study.
A.M., P.N., K.C.; Conceived and designed the
experiments. A.M.; Performed the experiments. A.M.,
P.N.; Analysed the data. All authors contributed to
reagents/materials/analysis tools and wrote the paper. All
authors read and approved the final manuscript.
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