Mutations In Transmembrane Domain Research Articles

Geospatial share of fungicide resistant Botrytis cinerea mutations in the Tokaj and Eger wine regions according to local pest management strategies

AbstractBotrytis cinerea is one of the fungal pathogens with the widest host plant spectrum, causing serious yield losses and significant economic damage in vineyards from year to year. As an ubiquitous, polyphagous fungal pathogen, with both saprophytic and parasitic lifestyle. The sequential use of active substances belonging to the same chemical family to protect vineyards can lead to an increase in fungal chemical resistance, which is reflected in the enrichment of point mutations in the genomic regions coding proteins involved in the mechanism of action of different pesticides. The aim of our studies was to compare the sensitivity to different fungicides of B. cinerea populations in two wine regions with different pest management practices: the Tokaj region, where the presence of B. cinerea is necessary to produce noble rot wines, and the adjacent Eger Region, where a total protection against B. cinerea is desired. Our study is the first Hungarian report of some previously studied resistance mutations in ERG27 and SDHB protein-coding genes. We identified point mutations in ERG27 transmembrane domain that have not been previously described but may affect the emergence of resistance to certain fungicides. Our study shows that the B. cinerea population of the Northern Hungary region is consistently characterized by an increase in fenhexamid resistance.

Divergent folding-mediated epistasis among unstable membrane protein variants.

Many membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for the mammalian gonadotropin-releasing hormone receptor GPCRs (GnRHR). We recently demonstrated that evolutionary GnRHR modifications appear to have coincided with adaptive changes in cotranslational folding efficiency. Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations. We therefore compared the pairwise interactions formed by mutations that disrupt the membrane topology (V276T) or tertiary structure (W107A) of GnRHR. Using deep mutational scanning, we evaluated how the plasma membrane expression of these variants is modified by hundreds of secondary mutations. An analysis of 251 mutants in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the severity and the mechanism of destabilization. V276T forms predominantly negative epistatic interactions with destabilizing mutations in soluble loops. In contrast, W107A forms positive interactions with mutations in both loops and transmembrane domains that reflect the diminishing impacts of the destabilizing mutations in variants that are already unstable. These findings reveal how epistasis is remodeled by conformational defects in membrane proteins and in unstable proteins more generally.

Divergent folding-mediated epistasis among unstable membrane protein variants

Many membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for the mammalian gonadotropin-releasing hormone receptor GPCRs (GnRHR). We recently demonstrated that evolutionary GnRHR modifications appear to have coincided with adaptive changes in cotranslational folding efficiency. Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations. We therefore compared the pairwise interactions formed by mutations that disrupt the membrane topology (V276T) or tertiary structure (W107A) of GnRHR. Using deep mutational scanning, we evaluated how the plasma membrane expression of these variants is modified by hundreds of secondary mutations. An analysis of 251 mutants in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the severity and the mechanism of destabilization. V276T forms predominantly negative epistatic interactions with destabilizing mutations in soluble loops. In contrast, W107A forms positive interactions with mutations in both loops and transmembrane domains that reflect the diminishing impacts of the destabilizing mutations in variants that are already unstable. These findings reveal how epistasis is remodeled by conformational defects in membrane proteins and in unstable proteins more generally.

Clinical and structural insights into the rare but oncogenic HER2-activating missense mutations in non-small cell lung cancer: a retrospective ATLAS cohort study

BackgroundUnlike human epidermal growth factor receptor 2 (HER2) amplification or exon 20 insertions, missense mutations in the extracellular domain (ECD), transmembrane domain (TMD), and intracellular domain (ICD) of the HER2 protein have been implicated as oncogenic in non-small cell lung cancer (NSCLC). However, their molecular subtypes, structural disparities, and clinical responses to current medical treatments, particularly HER2-targeted tyrosine kinase inhibitors (TKIs), remain unclear in NSCLC and warrant investigation.MethodsA real-world observational ATLAS study was conducted to gather and analyze therapeutic outcomes of chemotherapy or TKIs for heterogeneous HER2 missense mutations in NSCLC. Computational models of typical ECD, TMD, and ICD mutations were utilized to explore their structural variances.ResultsWe screened 37 eligible patients with HER2-activating missense mutations, of which 35 patients who had received chemotherapy or HER2-targeted TKIs as first-line therapy were available for response assessment. The median progression-free survival (PFS) for chemotherapy was 4.43 months (95% confidence interval [CI], 3.77–5.10), with an objective response rate (ORR) of 26.1% (6/23) and a disease control rate (DCR) of 17/23 (73.9%). The administration of afatinib, dacomitinib, and pyrotinib, HER2-targeted TKIs, achieved a median PFS of 4.65 months, with an ORR of 33.3% (4/12) and a DCR of 83.3% (10/12). Molecular modeling and computational simulations of ECD, TMD, and ICD mutations revealed their distinct structural characteristics.ConclusionIn comparison to chemotherapy, HER2-targeted TKIs demonstrated similar activity and PFS benefits for HER2-activating missense mutations in NSCLC.

Genomic characterization of Influenza A (H1N1)pdm09 and SARS-CoV-2 from Influenza Like Illness (ILI) and Severe Acute Respiratory Illness (SARI) cases reported between July–December, 2022

Influenza Like Illness (ILI) and Severe Acute Respiratory Infection (SARI) cases are more prone to Influenza and SARS-CoV-2 infection. Accordingly, we genetically characterized Influenza and SARS-CoV-2 in 633 ILI and SARI cases by rRT-PCR and WGS. ILI and SARI cases showed H1N1pdm09 prevalence of 20.9% and 23.2% respectively. 135 (21.3%) H1N1pdm09 and 23 (3.6%) H3N2 and 5 coinfection (0.78%) of H1N1pdm09 and SARS-CoV-2 were detected. Phylogenetic analysis revealed H1N1pdm09 resemblance to clade 6B.1A.5a.2 and their genetic relatedness to InfA/Perth/34/2020, InfA/Victoria/88/2020 and InfA/Victoria/2570/2019. Pan 24 HA and 26 NA nonsynonymous mutations and novel HA (G6D, Y7F, Y78H, P212L, G339R, T508K and S523T) and NA (S229A) mutations were observed. S74R, N129D, N156K, S162N, K163Q and S164T alter HA Cb and Sa antibody recognizing site. Similarly, M19T, V13T substitution and multiple mutations in transmembrane and NA head domain drive antigenic drift. SARS-CoV-2 strains genetically characterized to Omicron BA.2.75 lineage containing thirty nonsynonymous spike mutations exhibited enhanced virulence and transmission rates. Coinfection although detected very minimal, the mutational changes in H1N1pdm09 and SARS-CoV-2 virus infected individuals could alter antibody receptor binding sites, allowing the viruses to escape immune response resulting in better adaptability and transmission. Thus continuous genomic surveillance is required to tackle any future outbreak.

A novel IL2RG transmembrane mutation resulting in severe combined immunodeficiency and maternal T cell engraftment

IntroductionX-linked severe combined immunodeficiency (SCID) is commonly associated with mutations in IL2RG, an integral part of several receptors essential for lymphocyte development. In this case, our patient has a novel IL2RG missense mutation in the transmembrane domain leading to a T-B+NK+ phenotype. Case PresentationA 2-week-old boy born full-term from an uncomplicated, non-consanguineous pregnancy presents for absent T-cell receptor excision circles (TREC) on newborn screen. He had been growing well, no thrush/diarrhea/infections. Patient had an uncle who died at 1-year-old from pneumonia but had no other family history. On exam he was healthy appearing, no dysmorphic features, rash, or hepatosplenomegaly.CBC was notable for absolute lymphocyte count of 530. TBNK flow cytometry at 2-weeks-old revealed < 1% CD3+ cells/CD4+ helper cells/CD8+ cytotoxic T cells, B cells were normal at 410, NK cells were mildly low 148, CD45Ra was < 1%, and CD45Ro was 100% (Table 1). Mitogen stimulation was markedly decreased. Microarray was normal, without 22q11.2 deletion. Primary immunodeficiency panel revealed hemizygous variance at the IL2RG gene c.811G>A (p.Gly 271Arg) that is not seen in the general population.Patient was diagnosed with T-B+NK+ SCID. Mom was CMV+ and stopped breastfeeding. Patient was started on IVIG and prophylactic therapy: fluconazole, acyclovir, and bactrim. He was referred to BMTand enrolled in a gene therapy trial as he has no matched sibling donors. At 2-months-old, repeat TBNK was notable for increased CD3 and CD4 cells, raising the concern for maternal engraftment (Table 1). Subsequent HLA chimerism study of T cells demonstrated HLA from mother(CD3) 91% and HLA from patient(CD3) 9% indicating engraftment of maternal cells. Patient was asymptomatic, no diarrhea or rash.Table 1Labs2-weeks-old2-months-oldTBNKAbsolute CD3+ (%)1 (<1%)49 (3%)Absolute CD3+4+ (%)1 (<1%)47 (3%)Absolute CD3+CD8+(%)1 (<1%)1 (<1%)CD4CD8 Ratio233.67Absolute CD19+ (%)410 (72%)1611 (92%)Absolute CD56+16+ (%)148 (26%)79 (5%)CD45 Ra<1%CD45 Ro100%Mitogen StimulationPHA2988 cpmCon A3774 cpmPWM1654 cpm DiscussionThe IL2RG gene G271A mutation results in a missense mutation that changes the charge of the amino acid glycine(uncharged) to arginine(positively charged) at the transmembrane region of the gamma chain, which likely interferes with the receptor's localization on the cell surface. Interestingly, NK cells are preserved in this patient indicating some preservation of signaling by IL15. This case illustrates a novel mutation in the IL2RG transmembrane domain that results in T-B+NK+ phenotype with maternal engraftment.

Abstract 2524: Critical interactions and tumor-specific mutations of Bcl-2 transmembrane domains revealed by a novel split luciferase assay

Abstract In intrinsic apoptosis, the interaction network of the Bcl-2 protein family controls the decision over life and death. Cells are sentenced to death when pro-apoptotic multidomain effector proteins BAX, BAK or BOK oligomerize and form pores in the mitochondrial outer membrane. This releases cytochrome c, which induces the activation of cell-wrecking proteases, the caspases. Interactions of Bcl-2 proteins with other family members essentially regulate cell death. The interaction via the BH3 region was intensively studied in the last decades. As a result, small-molecule drugs, BH3-mimetics, were developed which bind and inhibit anti-apoptotic Bcl-2 proteins. Since anti-apoptotic Bcl-2 proteins are often overexpressed in hematopoietic malignancies, BH3-mimetics e.g. Venetoclax are approved for anti-cancer therapy. Unlike the BH3 interaction site, the c-terminal α9-helix referred to as the transmembrane domain (TMD) is mostly neglected in Bcl-2 interaction studies. However, TMDs not only dictate subcellular localization, but also substantially influence protein-protein interactions. Interestingly, Bcl-2 TMDs can harbor several tumor-specific mutations. The functional basis for TMD interaction as well as the resulting functional relevance for apoptosis signaling, however, remains poorly understood. To unravel the Bcl-2 TMD interaction network, we developed a split luciferase assay system enabling us to detect Bcl-2 TMD interactions in living cells. Simultaneously encoding for the expression of a fluorophore and TMD fusion peptides this system was used to generate fluorescence-normalized luminescence-based interaction data. Here, we identified a homotypic interaction pattern among effector TMDs of BAX, BAK and BOK. Molecular modelling of effector TMD interaction in mimics of cellular membranes also supports these findings. TMD swap experiments show significant influence of TMD sequence on subcellular localization and cell death signaling as assessed via confocal laser scanning microscopy and flow cytometry-based cell death assays. Moreover, we tested previously described mutations of the BAX-TMD (S184A, S184D) as well as a tumor-specific mutation (V180G) in the novel interaction assay. S184 (de-)phosphorylation as mimicked with S184A/S184D affects subcellular localization. In accordance, we find that S184A enhanced and S184D abolished interaction with wildtype BAX-TMD. Intriguingly, V180G not only modulates BAX subcellular localization but also prevents interaction with wildtype BAX-TMD. These findings verify a crucial role of Bcl-2 TMDs in subcellular localization and furthermore strongly support a function in interaction and cell death regulation. Further efforts to explore the Bcl-2 TMD interaction network as well as functional analysis of tumor-specific TMD mutations could pave the way to establish TMDs as a target of cancer therapy. Citation Format: Tobias B. Beigl, Alexander Paul, Sandra Weller, Benjamin Schäfer, Walter E. Aulitzky, Hans-Georg Kopp, Thomas Fellmeth, Kristyna Pluhackova, Markus Rehm, Frank Essmann. Critical interactions and tumor-specific mutations of Bcl-2 transmembrane domains revealed by a novel split luciferase assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2524.

Canonical Thrombopoietin Receptor Mutants in Myeloproliferative Neoplasms Rely Differently on the Extracellular Domain of the Receptor but Share Similar Mechanisms of Inactivation: An Open Field for the Development of New Therapies

Specific mutations in the thrombopoietin receptor (TpoR/MPL) are responsible for 2% of essential thrombocythemia (ET) and 5% of primary myelofibroses (PMF). The most prevalent oncogenic mutations, W515K/L/R/S/A and S505N, occur in the transmembrane domain (TMD) of TpoR. They lead to dimerization of TpoR in an active conformation in absence of its ligand, thrombopoietin (Tpo), and activation of downstream signaling mainly through the JAK/STAT pathway. We demonstrated the critical role of residue W491 in the extracellular juxtamembrane (JM) domain in the activation of TpoR through canonical S505N and W515K mutations and other non-canonical activating mutations (Levy G et al., Blood, 2020). We report here on the rare but recurrent insertion of the 4 residues VIAL between positions L498 and H499 ("insVIAL” hereunder) in ET patients. The mutation activated signaling in the absence of Tpo in luciferase and cell proliferation assays. We then asked whether the oncogenic TMD mutants of TpoR that induce MPNs depend exclusively on the transmembrane-intracellular (TM-IC) regions or also on the extracellular domain. To this end, we created a truncated TpoR that only contained the TM-IC regions. In luciferase assays, TM-IC TpoR was activated weakly by Elt, but not by Tpo nor calreticulin mutant del52, as expected, as both Tpo and CALR mutants require binding to the extracellular domain of TpoR. Furthermore, the W515K mutant was active in the TM-IC configuration, while S505N and insVIAL were not (Figure 1A). Longer constructs containing the full extracellular membrane-proximal fibronectin type III domain (D4) did not rescue activation by S505N or insVIAL, while there was some gain of activity with D3-D4 (D3 is the third fibronectin type III domain of TpoR). Of interest, D4 restored activation by S505N or insVIAL provided that H499 was mutated to leucine, the murine residue, which induces ligand-independent dimerization. Together, these results suggest that both intrachain structural changes and dimerization are required for activation, and that those can be accomplished by the W515 mutations but not by S505N or insVIAL alone. To explore differences further, we performed a cysteine (Cys) scanning mutagenesis in the JM-TM region and used the single Cys mutants on the background of a receptor with minimal number of Cys residues for o-PDM induced cross-linking. o-PDM induced different patterns of dimerization across mutants suggesting as well different mechanisms of activation. Using infrared spectroscopy, we also observed that W515K, H499L (which promotes helicity and dimerization in an inactive interface) or activation by Elt, all induce a common shift when compared to wild type JM-TM-JM domains, suggesting a modification of the structure of the JM-TM-JM domain in the same direction. Surprisingly, S505N induced a shift that is opposite (left shift), suggesting that its effect may differ from that of W515, which would be in agreement with the absolute requirement of S505N to contain the extracellular domain for activation. Of further interest, the unraveling on the cytosolic juxtamembrane domain was present by NMR for S505N, as it was for W515K. This suggests that events at the upper end of the TMD around H499 and W491 are key for activation. To identify upstream residues key for activation of S505N and W515K in D4, we performed an alanine-scanning of this domain. This resulted in the identification of another substitution on the same helical face as W491, which, like W491, was required for activation by S505N, W515K and insVIAL, but not for Elt (Figure 1B). Importantly we showed that when the interface we define is induced by a W491C mutation, the disulfide-bonded TpoR is constitutively active, suggesting that the alpha helix upstream W491 needs to rotate in the interface for the mutations S505N, W515K and insVIAL to induce activation. Altogether, these results allow a better understanding of activation mechanisms of TpoR canonical and rarer mutants, that would rely not only on the TMD but also on (D3 and) D4, which could have a role in stabilizing the interactions of the TMD helices and their relative angle to one another. As such, these results open the field to designing more easily new targeted therapies for the treatment of MPNs, as they could be active at the surface of the cell membrane. With a broadened vision even, they lead to a renewed understanding of cytokine type I mechanisms of activation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Disrupting PTPRJ transmembrane-mediated oligomerization counteracts oncogenic receptor tyrosine kinase FLT3 ITD.

The receptor protein tyrosine phosphatase (RPTP) PTPRJ (also known as DEP-1) has been identified as a negative regulator of the receptor tyrosine kinase FLT3 signalling in vitro. The inactivation of the PTPRJ gene in mice expressing the constitutively active, oncogenic receptor tyrosine kinase FLT3 ITD aggravated known features of leukaemogenesis, revealing PTPRJ's antagonistic role. FLT3 ITD mutations resulting in constitutively kinase activity and cell transformation frequently occur in patients with acute myeloid leukaemia (AML). Thus, in situ activation of PTPRJ could be used to abrogate oncogenic FLT3 signalling. The activity of PTPRJ is suppressed by homodimerization, which is mediated by transmembrane domain (TMD) interactions. Specific Glycine-to-Leucine mutations in the TMD disrupt oligomerization and inhibit the Epidermal Growth Factor Receptor (EGFR) and EGFR-driven cancer cell phenotypes. To study the effects of PTPRJ TMD mutant proteins on FLT3 ITD activity in cell lines, endogenous PTPRJ was inactivated and replaced by stable expression of PTPRJ TMD mutants. Autophosphorylation of wild-type and ITD-mutated FLT3 was diminished in AML cell lines expressing the PTPRJ TMD mutants compared to wild-type-expressing cells. This was accompanied by reduced FLT3-mediated global protein tyrosine phosphorylation and downstream signalling. Further, PTPRJ TMD mutant proteins impaired the proliferation and in vitro transformation of leukemic cells. Although PTPRJ's TMD mutant proteins showed impaired self-association, the specific phosphatase activity of immunoprecipitated proteins remained unchanged. In conclusion, this study demonstrates that the destabilization of PTPRJ TMD-mediated self-association increases the activity of PTPRJ in situ and impairs FLT3 activity and FLT3-driven cell phenotypes of AML cells. Thus, disrupting the oligomerization of PTPRJ in situ could prove a valuable therapeutic strategy to restrict oncogenic FLT3 activity in leukemic cells.

Mutation in Transmembrane Domain 8 of Human Urate Transporter 1 Disrupts Uric Acid Recognition and Transport.

Human urate transporter 1 (hURAT1) is the most pivotal therapeutic target for hyperuricemia. Due to a lack of crystal structure information, the atomic structure of URAT1 is not clearly understood. In this study, a multiple sequence alignment was performed, and K393, a positively charged residue in transmembrane domain (TMD) 8, was observed to be highly conserved in organic anion transporters (OATs). K393 was substituted with a positively, negatively, and neutrally charged amino acid via site-directed mutagenesis and then used to transfect HEK293 cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) analyses indicated that mutants of K393 showed mRNA and protein expression levels similar to those in the WT group. The nonpositively charged mutants K393A, K393D, and K393E eliminated 70–80% of 14C-uric acid transport capacity, while the K393H mutant showed slight and the K393R mutant showed no reduced transport capacity compared with the WT group. Binding assays indicated that K393A, K393D, and K393E conferred lowered uric acid binding affinity. As indicated by the Km and Vmax values obtained from saturation kinetic experiments, K393A, K393D, and K393E showed increased Km values, but K393R and K393H showed Km values similar to those in the WT group. K393 also contributed to a high affinity for benzbromarone (BM) interaction. The inhibitory effects of BM were partly abolished in K393 mutants, with increased IC50 values compared with the WT group. BM also exhibited weaker inhibitory effects on 14C-uric acid binding in K393R and K393H mutants. In an outward homology model of URAT1, K393 was located in the inner part of the transport tunnel, and further molecular docking analysis indicated that uric acid and BM showed possible hydrogen bonds with K393. Mutants K393R and K393H showed possible interactions with uric acid, and positive charges confer high affinity for uric acid as revealed by their surface electrostatic potential. In conclusion, our data provide evidence that K393 is an important residue for the recognition of uric acid or inhibitors by URAT1.

Role of S protein transmembrane domain mutations in the development of occult hepatitis B virus infection

ABSTRACT Occult HBV infection (OBI) is a special infection status during Hepatitis B virus (HBV) infection. The underlying mechanism of its occurrence remains unclear. This study conducted sequencing analysis on 104 OBI plasma samples and 524 HBsAg positive samples from 29 blood centres, and searched for high-frequency mutations in transmembrane domain (TMD) of S protein in the OBI population. Plasmids with TMD high-frequency mutations were constructed, in vivo and in vitro functional experiments were performed to investigate possible molecular mechanisms of OBI occurrence. We found 22 high-frequency TMD mutations in genotype B OBI strains. Among them, five mutations can lead to impairment of HBsAg secretion; seven mutations had accumulated intracellular HBsAg while extracellular HBsAg didn’t decrease compared to wildtype. This study chose C85R from TMD2, F220C, and F220Y from TMD4 for further exploration. Protein structure predication showed these three mutant HBsAg displayed changed hydrophilic properties and tended to accumulate in the phospholipid bilayer of cell membrane. Mutant HBsAg’s secretion disorder may induce OBI. On the other hand, V168A + V177A from TMD3 expressed increased HBsAg both in intracellular and extracellular levels. This mutation had most unstable natural conformation and may be inclined to transition into V177A or V168A + S174N + V177A. These three mutations were more prone to mixed infection, presenting a state of coexistence, thus approaching the impaired secretion pattern of OBI. This study demonstrated TMD mutations could contribute to the occurrence of OBI and provided a theoretical basis for OBI study and the functional cure of chronic hepatitis B virus infection.

Abstract 3981: Targeting SRP receptor-beta reduces protein glycosylation and cell growth in lung cancer

Abstract PURPOSE: Lung cancer is a major cause of morbidity and mortality world wide. Signaling receptors, adhesion molecules, and surface antigens that drive lung cancer progression are regulated by N-glycosylation, a post-translational protein modification initiated in the ER. Strategies for inhibition of N-glycosylation have recently been refined through the discovery of small molecules that partially disrupt glycan transfer. Here we screened for the target of a novel small molecule that reduces protein glycosylation with the goal of identifying new therapeutic targets for lung cancer. METHODS: Pooled results from chemical library HTS efforts identified hit compounds with activity. Counter-screening was performed in A549 cells using a fluorescence-based reporter (Halo1N) that detects aberrant N-glycosylation. The cellular targets for one class of inhibitors were evaluated by combining a whole-genome CRISPR-Cas9 screen with FACS sorting for loss of pharmacologic function. Significant changes in gRNA between populations was determined by MAGeCK analysis. The interaction between a compound and its target was determined by CETSA, and mechanism of action was verified by gene knockout and rescue experiments. Glycosylation status of glycoproteins were measured by western blot and cell compartment fluorescence. Predicted compound binding sites on the protein were mutated using site-directed mutagenesis. RESULTS: The lead tool compound (CP-9) has an IC50 in uM range and was used as a probe for target identification. CP-9 activates Halo-1N fluorescence, and whole genome CRISPR-Cas9 screening was used to identify gRNAs associated with loss of CP-9 activity. MAGeCK analysis demonstrated significant enrichment of genes associated with the translocon and ribosome (FDR P &amp;lt; 0.01) in the non-fluorescent population. We tested components of the signal recognition particle-receptor complex for direct interactions using the CETSA and demonstrate CP-9 binding to SR-β but not SR-α. Knockout experiments confirm that inhibition of glycosylation by CP-9 is dependent on SR-β expression, and CP-9’s effect on glycosylation and was rescued by SR-β re-expression. Mutated SR-β diminished CP-9’s effects on glycosylation and reduced SR-β thermal stabilization. CP-9 disrupted an interaction between SR-β/SR-α and the ribosome-translocon-OST complex suggesting a mechanism of action. Interestingly, a transmembrane domain mutant (ΔTM) of SR-β reduced the CP-9 effect and glycoprotein expression. ΔTM-SR-β also significantly reduced cell proliferation in A459 lung adenocarcinoma cells. CONCLUSION: CP-9 partially inhibits N-glycosylation by interfering with SR-β/SR-α and the translocon complex, leading to the disruption of protein import and glycosylation in the ER. Transmembrane domain mutants of SR-β reduce lung cancer cell growth and suggest this protein as a potential target for lung cancer drug development in the future. Citation Format: Chatchai Phoomak, Joseph N. Contessa. Targeting SRP receptor-beta reduces protein glycosylation and cell growth in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3981.

Characterization of CD20 expression loss as a mechanism of resistance to mosunetuzumab in patients with relapsed/refractory B-cell non-Hodgkin lymphomas.

7526 Background: Mosunetuzumab (M) is a bispecific antibody targeting CD20 and CD3 that redirects T cells to engage and eliminate malignant B cells being developed for relapsed or refractory (R/R) B-cell non-Hodgkin lymphomas (B-NHL). CD20 is an optimal target, with uniform expression across B-NHL histologies and minimal receptor turnover. We characterized CD20 loss as a potential mechanism of resistance to M in patients (pts) on a Phase I/II trial (NCT02500407) receiving M monotherapy for the treatment (tx) of R/R B-NHL. Methods: Pts with R/R B-NHL received M intravenously in 3-week cycles, for eight to 17 cycles depending on tumor response. At baseline (BL), biomarker-evaluable (archival or fresh) biopsies were collected from 293 pts. Biopsies from 62 pts were collected at additional time points during tx with M and/or at disease progression (PD). The proportion of CD20+ and PAX5+ tumor cells was determined by immunohistochemistry (IHC) using dual-staining with anti-CD20 (clone L26, VENTANA) and anti-PAX5 (clone DAK-PAX5, DAKO) antibodies. Expression of MS4A1, the gene encoding CD20 , was measured by RNA-sequencing (RNA-seq); MS4A1 mutation profiling was performed by whole exome sequencing (WES). Levels of CD20 expression were assessed relative to response rates. Correlative analyses were performed and assessed centrally (IHC, RNA-seq, and WES) and locally (IHC). Results: CD20 levels were consistently high ( &gt; 75% CD20+PAX5+ cells) in the majority of BL biopsies and generally comparable across histologies (FL, DLBCL, tFL, MCL, and RT). BL CD20 loss (≤5% CD20+PAX5+ cells) was seen in 16/293 pts (5.5%), more commonly in aggressive NHL, and responses to M were not seen in these pts. Among 62 pts with BL and on-tx/at-PD biopsies, BL CD20 levels were ≤5% in 7/62 pts (11%) (6/7 pts [86%] progressed before completing Cycle 2). CD20 levels were maintained in on-tx biopsies from 23/24 pts (96%). At PD, biopsies showed CD20 loss in 7/26 pts (27%). For five pts with BL, on-tx and at-PD biopsies, all pts maintained CD20 while on-tx and 1/5 pts (20%) had CD20 loss at PD. There was no clear association between CD20 reduction and histology. Data from 185 BL biopsies showed generally concordant levels of CD20 gene and protein expression (r = 0.72). In 10/185 pts (5%), MS4A1 was expressed without detectable CD20 protein expression; DNA sequencing revealed novel mutations in MS4A1, including mutations leading to truncation of the protein. CD20 transmembrane and extra-cellular domain mutations were also observed but do not block CD20 expression. Conclusions: In pts with R/R B-NHL treated with M, low BL CD20 expression is associated with lack of response to M. During M tx, loss of tumor cell expression of CD20 is one mechanism of acquired resistance; however, CD20 expression is maintained in most pts with PD, implying alternative mechanisms for acquired M resistance. Clinical trial information: NCT02500407.

Syntaxin-1A modulates vesicle fusion in mammalian neurons via juxtamembrane domain dependent palmitoylation of its transmembrane domain.

SNAREs are undoubtedly one of the core elements of synaptic transmission. Contrary to the well characterized function of their SNARE domains bringing the plasma and vesicular membranes together, the level of contribution of their juxtamembrane domain (JMD) and the transmembrane domain (TMD) to the vesicle fusion is still under debate. To elucidate this issue, we analyzed three groups of STX1A mutations in cultured mouse hippocampal neurons: (1) elongation of STX1A's JMD by three amino acid insertions in the junction of SNARE-JMD or JMD-TMD; (2) charge reversal mutations in STX1A's JMD; and (3) palmitoylation deficiency mutations in STX1A's TMD. We found that both JMD elongations and charge reversal mutations have position-dependent differential effects on Ca2+-evoked and spontaneous neurotransmitter release. Importantly, we show that STX1A's JMD regulates the palmitoylation of STX1A's TMD and loss of STX1A palmitoylation either through charge reversal mutation K260E or by loss of TMD cysteines inhibits spontaneous vesicle fusion. Interestingly, the retinal ribbon specific STX3B has a glutamate in the position corresponding to the K260E mutation in STX1A and mutating it with E259K acts as a molecular on-switch. Furthermore, palmitoylation of post-synaptic STX3A can be induced by the exchange of its JMD with STX1A's JMD together with the incorporation of two cysteines into its TMD. Forced palmitoylation of STX3A dramatically enhances spontaneous vesicle fusion suggesting that STX1A regulates spontaneous release through two distinct mechanisms: one through the C-terminal half of its SNARE domain and the other through the palmitoylation of its TMD.

Whole genome sequence analysis showing unique SARS-CoV-2 lineages of B.1.524 and AU.2 in Malaysia.

SARS-CoV-2 has spread throughout the world since its discovery in China, and Malaysia is no exception. WGS has been a crucial approach in studying the evolution and genetic diversity of SARS-CoV-2 in the ongoing pandemic. Despite considerable number of SARS-CoV-2 genome sequences have been submitted to GISAID and NCBI databases, there is still scarcity of data from Malaysia. This study aims to report new Malaysian lineages of the virus, responsible for the sustained spikes in COVID-19 cases during the third wave of the pandemic. Patients with nasopharyngeal and/or oropharyngeal swabs confirmed COVID-19 positive by real-time RT-PCR with CT value < 25 were chosen for WGS. The selected SARS-CoV-2 isolates were then sequenced, characterized and analyzed along with 986 sequences of the dominant lineages of D614G variants currently circulating throughout Malaysia. The prevalence of clade GH and G formed strong ground for the presence of two Malaysian lineages of AU.2 and B.1.524 that has caused sustained spikes of cases in the country. Statistical analysis on the association of gender and age group with Malaysian lineages revealed a significant association (p <0.05). Phylogenetic analysis revealed dispersion of 41 lineages, of these, 22 lineages are still active. Mutational analysis showed presence of unique G1223C missense mutation in transmembrane domain of the spike protein. For better understanding of the SARS-CoV-2 evolution in Malaysia especially with reference to the reported lineages, large scale studies based on WGS are warranted.

A positive genetic selection for transmembrane domain mutations in HRD1 underscores the importance of Hrd1 complex integrity during ERAD.

Misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for ubiquitination and degradation by the proteasome. During this process, known as ER-associated degradation (ERAD), the ER-embedded Hrd1 ubiquitin ligase plays a central role in recognizing, ubiquitinating, and retrotranslocating scores of lumenal and integral membrane proteins. To better define the mechanisms underlying Hrd1 function in Saccharomyces cerevisiae, several model substrates have been developed. One substrate is Sec61-2, a temperature sensitive allele of the Sec61 translocation channel. Cells expressing Sec61-2 grow at 25°C because the protein is stable, but sec61-2 yeast are inviable at 38°C because the mutated protein is degraded in a Hrd1-dependent manner. Therefore, deleting HRD1 stabilizes Sec61-2 and hence sec61-2hrd1∆ double mutants are viable at 38°C. This unique phenotype allowed us to perform a non-biased screen for loss-of-function alleles in HRD1. Based on its importance in mediating substrate retrotranslocation, the screen was also developed to focus on mutations in sequences encoding Hrd1's transmembrane-rich domain. Ultimately, a group of recessive mutations was identified in HRD1, including an ensemble of destabilizing mutations that resulted in the delivery of Hrd1 to the ERAD pathway. A more stable mutant resided in a buried transmembrane domain, yet the Hrd1 complex was disrupted in yeast expressing this mutant. Together, these data confirm the importance of Hrd1 complex integrity during ERAD, suggest that allosteric interactions between transmembrane domains regulate Hrd1 complex formation, and provide the field with new tools to define the dynamic interactions between ERAD components during substrate retrotranslocation.

Neurotrophin Receptor Kinase: Updated Perspectives, Laboratory Considerations, and Clinical Implications

Neurotrophin Receptor Kinase: Updated Perspectives, Laboratory Considerations, and Clinical Implications

SLC25A38 congenital sideroblastic anemia: Phenotypes and genotypes of 31 individuals from 24 families, including 11 novel mutations, and a review of the literature.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited disorders of erythropoiesis characterized by pathologic deposits of iron in the mitochondria of developing erythroblasts. Mutations in the mitochondrial glycine carrier SLC25A38 cause the most common recessive form of CSA. Nonetheless, the disease is still rare, there being fewer than 70 reported families. Here we describe the clinical phenotype and genotypes of 31 individuals from 24 families, including 11 novel mutations. We also review the spectrum of reported mutations and genotypes associated with the disease, describe the unique localization of missense mutations in transmembrane domains and account for the presence of several alleles in different populations.

Syndecan Transmembrane Domain Specifically Regulates Downstream Signaling Events of the Transmembrane Receptor Cytoplasmic Domain.

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

A Novel Probable Pathogenic PSEN2 Mutation p.Phe369Ser Associated With Early-Onset Alzheimer's Disease in a Chinese Han Family: A Case Report

The pathogenesis of Alzheimer's disease is complex, and early-onset Alzheimer's disease (EOAD) is mostly influenced by genetic factors. Presenilin-1, presenilin-2 (PSEN2), and amyloid precursor protein are currently known as the three main causative genes for autosomal dominant EOAD, with the PSEN2 mutation being the rarest. In this study, we reported a 56-year-old Chinese Han proband who presented with prominent progressive amnesia, aphasia, executive function impairment, and depression 5 years ago. The 3-year follow-up showed that the patient experienced progressive brain atrophy displayed on magnetic resonance imaging (MRI) and dramatic cognitive decline assessed by neuropsychological evaluation. This patient was clinically diagnosed as EOAD based on established criteria. A heterozygous variant (NM_000447.2: c.1106T>C) of PSEN2 was identified for the first time in this patient and her two daughters. This mutation causing a novel missense mutation (p.Phe369Ser) in transmembrane domain 7 encoded by exon 11 had not been reported previously in 1000Genomes, ExAC, or ClinVar databases. This mutation was predicted by four in silico prediction programs, which all strongly suggested that it was damaging. Our results suggest that this novel PSEN2 Phe369Ser mutation may alter PSEN2 protein function and associate with EOAD.