Deregulation of LRSAM1 expression impairs the levels of TSG101, UBE2N, VPS28, MDM2 and EGFR

CMT is the most common hereditary neuromuscular disorder of the peripheral nervous system with a prevalence of 1/2500 individuals and it is caused by mutations in more than 80 genes. LRSAM1, a RING finger ubiquitin ligase also known as TSG101-associated ligase (TAL), has been associated with Charcot-Marie-Tooth disease type 2P (CMT2P) and to date eight causative mutations have been identified. Little is currently known on the pathogenetic mechanisms that lead to the disease. We investigated the effect of LRSAM1 deregulation on possible LRSAM1 interacting molecules in cell based models. Possible LRSAM1 interacting molecules were identified using protein-protein interaction databases and literature data. Expression analysis of these molecules was performed in both CMT2P patient and control lymphoblastoid cell lines as well as in LRSAM1 and TSG101 downregulated SH-SY5Y cells.TSG101, UBE2N, VPS28, EGFR and MDM2 levels were significantly decreased in the CMT2P patient lymphoblastoid cell line as well as in LRSAM1 downregulated cells. TSG101 downregulation had a significant effect only on the expression of VPS28 and MDM2 and it did not affect the levels of LRSAM1. This study confirms that LRSAM1 is a regulator of TSG101 expression. Furthermore, deregulation of LRSAM1 significantly affects the levels of UBE2N, VPS28, EGFR and MDM2.


Introduction 47
Charcot-Marie-Tooth (CMT) disease is the most common inherited neuropathy, characterized by 48 peripheral nervous system degeneration, with a relatively high frequency (1:2,500 individuals) (1, 49 2). CMT disease is classified into demyelinating, axonal and intermediate types based on 50 electrophysiological findings (3). It is further sub-divided into an increasing number of genetic 51 subtypes with more than 80 CMT genes currently known. 52 Leucine Rich Repeat And Sterile Alpha Motif Containing 1 (LRSAM1) has been associated with 53 Charcot-Marie-Tooth disease type 2P (CMT2P). LRSAM1, is a RING finger ubiquitin ligase, 54 which participates in a range of functions, including cell adhesion, signaling pathways and cargo 55 sorting through receptor endocytosis (4, 5). It is a multidomain protein with an N-terminal 56 leucine-rich repeat (LRR) domain, followed by an Ezrin-Radixin-Moezin (ERM) domain, a 57 coiled-coil (CC) region, a SAM domain, a PSAP/PTAP tetrapeptide motif and a RING finger 58 domain (6). LRSAM1, also known as TAL (TSG101-associated ligase), regulates the metabolism 59 of TSG101 (Tumor Susceptibility gene 101) by polyubiquitination (7). TSG101 is a tumor 60 suppressor gene component of the ESCRT machinery (Endosomal Sorting Complex Required for 61 Transport), with a significant role in cell cycle regulation and differentiation (5,8). LRSAM1 62 binds to TSG101, through a bivalent binding of the PTAP motif and the CC domain of LRSAM1 63 (5,9). After binding, LRSAM1 attaches several monomeric ubiquitins to the C-Terminal of 64 TSG101, thus regulating its function. LRSAM1 mutations impair the LRSAM1-TSG101 65 interaction (10). 66 We identified a dominant LRSAM1 mutation (c.2047-1G>A, p.Ala683ProfsX3) located in the 67 RING finger domain of LRSAM1 (11). Recently, we reported that downregulation of LRSAM1 68 affects the proliferation and morphology of neuroblastoma SH-SY5Y cells, and, overexpression 69 of wild-type LRSAM1 rescues, while the c.2047-1G>A mutant fails to rescue the phenotype of the based evaluation these molecules were selected for further analysis. Furthermore, the AP-3 was 95 selected for analysis since in a recent study LRSAM1 immunoprecipitated with . 96

RNA expression analysis of interacting molecules in lymphoblastoid cell lines 98
We investigated RNA expression of candidate interacting molecules (TSG101,UBE2N,VPS28,99 EGFR, in the CMT2P patient and control lymphoblastoid cell lines. mRNA 100 levels of TSG101, UBE2N, VPS28, MDM2 and EGFR were significantly reduced in the patient 101 lymphoblastoid cell line as compared to the control, while AP-3 presented an equal expression in 102 both patient and control lymphoblastoid cell lines. Specifically, the patient sample showed 103 significantly lower mRNA levels of TSG101 (43%), UBE2N (45%), VPS28 (32%), EGFR (41%) 104 and MDM2 (35%) as compared to the control sample. In contrast AP-3 (98%) showed a similar 105 expression between the patient and control samples ( Figure 4) Figure 5). Although, LRSAM1 downregulation caused a significant reduction of 120 TSG101 levels (55%), TSG101 downregulation did not affect the expression of LRSAM1. 121

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LRSAM1 has been associated with CMT disease and currently its role in neurodegenerative 123 disorders and in CMT pathogenesis is still unclear. In the present study we found that 124 deregulation of LRSAM1 impaired TSG101, UBE2N, VPS28, MDM2 and EGFR levels. TSG101 125 downregulation had an effect on VPS28 and MDM2 levels. 126 The E3 ligase activity of LRSAM1 performs ubiquitination of TSG101, regulating its expression 127 (5). TSG101 levels are also modulated by other mechanisms beyond LRSAM1 E3 regulation. 128 MDM2, another E3 ligase, shares an autoregulatory loop with TSG101 (15). TSG101 prevents its 129 ubiquitination and increases the levels of MDM2 thus promoting ubiquitin degradation of p53 130 (15, 16). Over expression of truncated TSG101 in U20S cells caused reduction of MDM2 levels 131 (15), while over expression of normal TSG101 doubled the short half-life of MDM2 and elevated 132 its expression levels (15). Notably we detected that 70% downregulation of LRSAM1 caused 133 55% decrease in TSG101 and also 55% decrease in MDM2, whereas 70% downregulation of 134 TSG101, which did not affect LRSAM1, caused only 20% decrease in MDM2 expression. These 135 results indicate that TSG101 exerts a small effect on MDM2 expression (20%) relative to the 136 effect of LRSAM1 (55%), the later through an unknown mechanism. 137 Interaction of TSG101 (yeast VPS23) with VPS28, VPS37 and MVB12 is necessary for the 138 formation of the ESCRT-I complex (17). Binding between TSG101 and ESCRT-I subunits 139 protects TSG101 degradation (9). VPS28 binds to the C-Terminal of TSG101 and blocks the 140 interaction with LRSAM1, thus subsequently decreases the ubiquitination of TSG101 (18). 141 Reduced levels of VPS28 were observed in cells that lack TSG101 (18), while over expression of 142 VPS28 did not affect the levels of TSG101 (9). In our study, we observed decreased levels of VPS28 in the CMT2P patient lymphoblastoid cell line and showed that down regulation of 144 LRSAM1 or TSG101 caused a significant reduction in VPS28 levels. It is thus possible that 145 mutant or knockdown LRSAM1 affects VPS28 levels directly or through the impaired TSG101 146 levels. TSG101 and/or VSP28 deficiency possibly affect the formation of the ESCRT-I complex. We observed a decreased expression of UBE2N in the patient lymphoblastoid cell line and also in 160 LRSAM1 downregulated cells. A recent study showed, using a ubiquitination assay, that 161 LRSAM1 interacts with UBC13, a mouse homolog of UBE2N and that mutations of LRSAM1 162 prevent this interaction, abolishing the ubiquitination activity of LRSAM1(10). UBE2N belongs 163 to the family of Ubiquitin-conjugating enzymes (E2) and it is the only known enzyme that 164 facilitates the creation of K63-linked polyubiquitination chains on proteins which are selected for 165 proteasomal degradation (22, 23). E2 along with E1 and E3 enzymes consist the UPS system 166 which removes the misfolded and abnormal proteins within the cells. Alzheimer, Parkinson and 167 Huntington neurodegenerative diseases are caused by the formation of inclusions in the brain because of the accumulation of misfolded proteins in neurons (23), indicating a possible 169 pathogenic role of the UPS system in neurodegeneration. Expression of UBE2N was found to be 170 elevated in synaptosomes in tissues obtained from aged monkey brain while UPS activity was 171 found to be depressed. The increased level of UBE2N in aged monkey brain or the over 172 expressed UBE2N in HEK293 cells, caused the accumulation of mutant huntingtin (HTT). In 173 contrast, the decreased level of UBE2N in aged monkey brain, HEK293 and PC12 cells caused The RING finger domain of LRSAM1 also plays a role in the regulation of EGFR expression (5). 178 We observed that the LRSAM1 c.2047-1G>A mutation, located in the RING finger domain, 179 results in impaired EGFR expression. Transfection of an inactive mutant-RING LRSAM1 has 180 been reported to lead to the reduction of EGFR expression, while transfection of wild type 181 LRSAM1 improved EGFR expression (5). Also in the presence of mutant TSG101 in NIH 3T3 182 and SL6 cells, EGFR moved from the early endosomes and MVBs to the plasma membrane for 183 recycling (25). In our study, downregulation of TSG101 did not affect the expression of EGFR. 184 The EGF receptor is a family member of the ErbB receptors of kinases and regulates essential 185 signaling pathways including cell proliferation and differentiation, cell cycle and migration (26). 186 Upregulation of EGFR has been associated with both oncogenesis and neurodegeneration. 187 Expression of EGFR is activated in many malignancies such as breast cancer, prostate cancer and 188 gliomas (27)  AP-3 has been reported to form a heterotetrameric complex, targeting cargos of lysosomes and 193 lysosome related organelles (29). Recently LRSAM1 was identified as an 194 because it immunoprecipitated with AP-3. It has been proposed, that while LRSAM1 is bound to 195 AP-3, it regulates the ESCRT-I sorting function, by ubiquitinating certain cargoes (14). We found 196 normal expression of AP-3 in the CMT2P patient lymphoblastoid cell line as well as after 197 LRSAM1 downregulation in SH-SY5Y cells. These data, suggest that the interaction between the 198 two molecules is probably controlled by AP-3 and not LRSAM1. 199 In conclusion, we confirm that TSG101 expression is regulated by LRSAM1. Decreased levels of 200 UBE2N and EGFR were observed only after LRSAM1 knockdown thus indicating that these two 201 molecules are regulated by a mechanism in which only LRSAM1 is involved. Decreased 202 expression of VPS28 and MDM2 after either LRSAM1 or TSG101 downregulation demonstrates 203 that the levels of these two molecules are regulated by a mechanism in which both LRSAM1 and 204 TSG101 may be involved. Further investigation of the above molecules may enable delineation of 205 the pathways that lead to CMT2P pathology. 206

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In this study, we selected possible LRSAM1 interacting molecules and investigated their 208 expression levels in lymphoblastoid cell lines as well as in LRSAM1 and TSG101 downregulated 209 neuroblastoma SH-SY5Y cells.

Protein extraction from experimental SH-SY5Y cells and lymphoblastoid cell lines 268
Proteins were extracted using a protein lysis buffer, that contained 1M NaCl, 10mM Tris-Cl 269

Statistical analysis 294
Quantitative data (ratio %) from three independent experiments were analyzed using the two-295 tailed Student's paired t-test. Thresholds of P-value < 0.05 were considered statistically 296 significant. All the data are expressed as the mean% ± standard deviation (SD) from three 297 independent experiments. The mean of quantitative data of the control samples was set to 100%.

Conflict of Interest Statement 303
The authors declare no conflict of interest.