Recombinant Human Transmembrane protein 59-like (TMEM59L)

What is the basic structure and cellular localization of TMEM59L?

TMEM59L is a type I transmembrane protein with high conservation across species (75% identity between human and mouse TMEM59L). It contains a single potential N-linked glycosylation site (N97 in TMEM59L, compared to N90 in TMEM59) that undergoes modest N-glycosylation . Subcellular localization studies show that TMEM59L is partially colocalized with early endosome marker Rab5, late endosome marker Rab7, recycling endosome marker Rab11, and cis-Golgi marker Giantin . Its localization to vesicular structures in the Golgi bodies and cell membrane can be observed in living cells . Unlike its homolog TMEM59, which is ubiquitously expressed, TMEM59L expression is predominantly limited to neurons .

How does TMEM59L expression change during development?

TMEM59L expression has a distinct developmental pattern. It is detected in the brain as early as embryonic day 8.5 (E8.5) and increases progressively until 3 months of age . In contrast, TMEM59 expression remains relatively low and stable throughout different developmental stages . This temporal expression pattern suggests TMEM59L may play important roles in neural development and maturation processes .

What are the best methods for overexpressing or knocking down TMEM59L in research models?

For TMEM59L overexpression, lentiviral delivery systems have proven effective. Lentivirus expressing TMEM59L (Lenti-TMEM59, typically tagged with Flag) can be bilaterally delivered into specific brain regions, such as the hippocampus . For RNA interference, shRNA targeting mouse Tmem59l can be constructed in pLL3.7 vector with validated sequences such as 5′-GCTATCCTGATCAGTGCTT-3′ (shRNA1) and 5′-GAGAGTGACTTCCTCAGTT-3′ (shRNA2) . For in vivo applications, these shRNA sequences can be inserted into pAKD-CMV-bGlobin-eGFP-H1-shRNA vector for adeno-associated virus (AAV) packaging . Expression verification should be performed using Western blotting or immunofluorescence analyses with appropriate antibodies.

How can researchers effectively produce and purify recombinant TMEM59L protein?

Recombinant TMEM59L protein can be produced in E. coli expression systems with an N-terminal His6-ABP tag or similar affinity tags . The protein is typically purified by IMAC chromatography with expected concentrations greater than 0.5 mg/ml . For storage, PBS with 1M Urea at pH 7.4 is recommended, with storage at -20°C while avoiding freeze-thaw cycles . Quality control should include SDS-PAGE with Coomassie blue staining to confirm >80% purity . Researchers should note that recombinant TMEM59L is often produced as a partial protein corresponding to specific domains rather than the full-length protein due to the challenges in expressing transmembrane proteins.

How does TMEM59L contribute to neuronal death in oxidative stress conditions?

TMEM59L plays a significant role in mediating oxidative stress-induced neuronal death through multiple pathways. Upon hydrogen peroxide exposure, TMEM59L contributes to intrinsic caspase-dependent apoptosis . Mechanistically, TMEM59L overexpression induces autophagy through interaction with ATG5 and ATG16L1, but concurrently triggers apoptotic pathways more dramatically than its homolog TMEM59 . Importantly, downregulation of TMEM59L can prevent neuronal cell death and caspase-3 activation caused by hydrogen peroxide insults and reduce the lipidation of LC3B . This protective effect suggests TMEM59L serves as a critical mediator that connects oxidative stress signals to cell death execution in neurons.

What is the role of TMEM59L in Alzheimer's disease pathogenesis?

TMEM59L significantly influences Alzheimer's disease pathology through multiple mechanisms. It modulates complex N- and O-glycosylation steps occurring during the Golgi maturation of amyloid precursor protein (APP), thereby inhibiting APP transport to the cell surface and further shedding . Overexpression of TMEM59L impairs short-term working memory in wild-type mice, suggesting its neurotoxic role . In the 5xFAD mouse model of Alzheimer's disease, TMEM59L overexpression exacerbates AD-like pathologies by increasing levels of detergent-insoluble Aβ and Aβ plaques, as well as dystrophic neurites . Conversely, haploinsufficiency of TMEM59 (the homolog of TMEM59L) reduced insoluble Aβ levels, Aβ plaques, and neurite dystrophy, thereby rescuing synaptic plasticity and memory deficits in 5xFAD mice .

What is the relationship between TMEM59L expression and immune microenvironment in cancer?

TMEM59L expression significantly correlates with the tumor immune microenvironment across multiple cancer types. Analysis shows that TMEM59L expression negatively correlates with activated CD4 T cells and CD8 T cells in most cancer types . Further immunophenoscore (IPS) analysis reveals that TMEM59L expression is negatively related to IPS score, Average Z-score (AZ), and Effector cells (ECs), while being positively associated with Suppressor cells (SCs) . TMEM59L expression is also negatively linked with the expression of many immune modulators, including PD-L1, IDO1, TIGIT, CTLA-4, and BTLA in various cancers . In the IMvigor210 cohort, high expression of TMEM59L correlates with poor clinical response to immune checkpoint blockade therapy, suggesting TMEM59L may contribute to an "immune-excluded" tumor microenvironment . These findings indicate TMEM59L may serve as a potential novel immune target.

How do TMEM59L and TMEM59 differ in their autophagy-inducing mechanisms?

Both TMEM59L and TMEM59 induce autophagy through interaction with ATG5 and ATG16L1, leading to LC3 lipidation, but with distinct potency and outcomes . TMEM59 contains a minimal 19-amino-acid peptide in its intracellular domain that promotes LC3 labelling and initiates the autophagic process . This subdomain (amino acids 263-281) contains a specific motif: [YW]-X₃-[ED]-X₄-[YWF]-X₂-L, which is required for promoting a functional complex between LC3 and ATG16L1 . While both proteins trigger autophagy, TMEM59L overexpression induces intrinsic caspase-dependent apoptosis more dramatically than TMEM59 . This differential effect on cell death pathways suggests that despite their shared ability to induce autophagy, TMEM59L and TMEM59 exhibit distinct regulatory mechanisms on downstream pathways that determine cell fate.

What signaling pathways are regulated by TMEM59L in cancer progression?

TMEM59L regulates multiple cancer-associated signaling pathways. Gene set enrichment analysis (GSEA) reveals that TMEM59L is involved in regulating immune pathways such as IL6-JAK-STAT3, IL2-STAT5, and TGF-β signaling . In various cancer types, TMEM59L expression correlates with pathway activity scores (PAS) of critical cancer-related pathways including apoptosis, cell cycle, DNA damage response, epithelial–mesenchymal transition (EMT), hormone androgen receptor (AR), hormone estrogen receptor (ER), TSC–mTOR, receptor tyrosine kinase (RTK), Ras/MAPK, and PI3K/AKT signaling pathways . The relationship between TMEM59L and these pathways varies across cancer types, with either activating or inhibitory effects depending on the specific cancer context. Additionally, TMEM59L expression negatively correlates with tumor mutational burden (TMB) in many cancers including HNSC, LUAD, LIHC, KIRC, BRCA, THCA, BLCA, KIRP, LGG, ESCA, PAAD, UCEC, and STAD, suggesting a potential role in regulating genomic stability .

How can TMEM59L be targeted therapeutically in neurological disorders and cancer?

Therapeutic targeting of TMEM59L represents a promising strategy based on its differential roles in neurological disorders and cancer. For neurological disorders, particularly those involving oxidative stress-induced neuronal death, downregulation of TMEM59L using RNA interference approaches has shown protective effects by preventing neuronal cell death and caspase-3 activation . In Alzheimer's disease models, targeting TMEM59L could potentially reduce Aβ accumulation by modulating APP processing and trafficking . For cancer applications, the approach would likely be context-dependent. In cancers where high TMEM59L expression correlates with poor prognosis, inhibition strategies using antibodies or small molecules might be beneficial . Given TMEM59L's role in creating an immunosuppressive microenvironment, combination approaches using anti-TMEM59L antibodies alongside other therapeutic interventions, particularly immune checkpoint inhibitors, may represent an effective strategy to enhance immunotherapy responses . Validation of these approaches requires development of specific inhibitors or activators of TMEM59L and testing in appropriate disease models.