An ERAD-independent role for rhomboid pseudoprotease Dfm1 in mediating sphingolipid homeostasis.

Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER), where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) removes these client proteins from the ER membrane to the cytosol in a process known as retrotranslocation. Our previous work demonstrated that rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated membrane integral ERAD substrates. Herein, we found that Dfm1 associates with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components that are critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. Furthermore, Dfm1 employs an ERAD-independent role for facilitating the ER export and endosome- and Golgi-associated degradation (EGAD) of Orm2, which is a major antagonist of SPT activity. Given that the accumulation of human Orm2 homologs, ORMDLs, is associated with various pathologies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.

Upregulation of sodium iodide symporter (NIS) protein expression by an innate immunity component: Promising potential for targeting radiosensitive retinoblastoma.

Retinoblastoma (RB), a malignant tumour of the eye arising from developing retina, is the most frequent primary intraocular malignancy of childhood. Its primary management with chemotherapy involves combination regimen of etoposide, vincristine and carboplatin and intra vitreal chemotherapy using melphalan when vitreous seeds develop. Radiotherapy is another effective mode in treating RB. We recently explored the notion if radiotherapy in RB can be mediated via Sodium Iodide Symporter (NIS), an intrinsic membrane glycoprotein which is a key regulator of iodide access to thyroid gland. Its expression has been exploited successfully for diagnostic imaging and molecular radionuclide-based therapy of thyroid cancer. We determined that NIS is expressed endogenously in RB tumour tissues, and in retinoblastoma cell lines Y79 and Weri-Rb-1, and therefore made an attempt to enhance the endogenously low expression of NIS protein in both Y79 and Weri-Rb-1 cells. Here we report about the potential of bovine lactoferrin (bLf) which is a known chemo preventive and emerging safe anti-cancer bio drug, as well as a natural transcriptional activator of genes, to enhance the endogenous expression of NIS in Y79 and Weri-Rb-1 cells. Real time PCR revealed that both cell lines express mRNA of lactoferrin receptors while flow cytometry and confocal microscopy showed the cells efficiently internalize bLf which upregulates NIS expression. These findings highlight an important step that could be taken towards the development of less harmful approaches for the treatment of RB by employing natural supplement bLf (with its clinically proven safe profile), and warrants further studies in future, focussing on enhancing NIS expression in RB cells and NIS functional assays in these cells.

Derlin rhomboid pseudoproteases employ substrate engagement and lipid distortion to enable the retrotranslocation of ERAD membrane substrates.

Nearly one-third of proteins are initially targeted to the endoplasmic reticulum (ER) membrane, where they are correctly folded and then delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) moves these clients from the ER membrane to the cytosol, a process known as retrotranslocation. Our recent work in Saccharomyces cerevisiae reveals a derlin rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated ERAD membrane substrates. In this study, we identify conserved residues of Dfm1 that are critical for retrotranslocation. We find several retrotranslocation-deficient Loop 1 mutants that display impaired binding to membrane substrates. Furthermore, Dfm1 possesses lipid thinning function to facilitate in the removal of ER membrane substrates, and this feature is conserved in its human homolog, Derlin-1, further implicating that derlin-mediated retrotranslocation is a well-conserved process.