Joseph D. Mancias

Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy

Autophagy, the process by which proteins and organelles are sequestered in double-membrane structures called autophagosomes and delivered to lysosomes for degradation, is critical in diseases such as cancer and neurodegeneration. Much of our understanding of this process has emerged from analysis of bulk cytoplasmic autophagy, but our understanding of how specific cargo, including organelles, proteins or intracellular pathogens, are targeted for selective autophagy is limited. Here we use quantitative proteomics to identify a cohort of novel and known autophagosome-enriched proteins in human cells, including cargo receptors. Like known cargo receptors, nuclear receptor coactivator 4 (NCOA4) was highly enriched in autophagosomes, and associated with ATG8 proteins that recruit cargo–receptor complexes into autophagosomes. Unbiased identification of NCOA4-associated proteins revealed ferritin heavy and light chains, components of an iron-filled cage structure that protects cells from reactive iron species but is degraded via autophagy to release iron through an unknown mechanism. We found that delivery of ferritin to lysosomes required NCOA4, and an inability of NCOA4-deficient cells to degrade ferritin led to decreased bioavailable intracellular iron. This work identifies NCOA4 as a selective cargo receptor for autophagic turnover of ferritin (ferritinophagy), which is critical for iron homeostasis, and provides a resource for further dissection of autophagosomal cargo–receptor connectivity.

Structure and Organization of Coat Proteins in the COPII Cage.

COPII-coated vesicles export newly synthesized proteins from the endoplasmic reticulum. The COPII coat consists of the Sec23/24-Sar1 complex that selects cargo and the Sec13/31 assembly unit that can polymerize into an octahedral cage and deform the membrane into a bud. Crystallographic analysis of the assembly unit reveals a 28 nm long rod comprising a central alpha-solenoid dimer capped by two beta-propeller domains at each end. We construct a molecular model of the COPII cage by fitting Sec13/31 crystal structures into a recently determined electron microscopy density map. The vertex geometry involves four copies of the Sec31 beta-propeller that converge through their axial ends; there is no interdigitation of assembly units of the kind seen in clathrin cages. We also propose that the assembly unit has a central hinge-an arrangement of interlocked alpha-solenoids-about which it can bend to adapt to cages of variable curvature.

KRAS: feeding pancreatic cancer proliferation

Oncogenic KRAS mutation is the signature genetic event in the progression and growth of pancreatic ductal adenocarcinoma (PDAC), an almost universally fatal disease. Although it has been appreciated for some time that nearly 95% of PDAC harbor mutationally activated KRAS, to date no effective treatments that target this mutant protein have reached the clinic. A number of studies have shown that oncogenic KRAS plays a central role in controlling tumor metabolism by orchestrating multiple metabolic changes including stimulation of glucose uptake, differential channeling of glucose intermediates, reprogrammed glutamine metabolism, increased autophagy, and macropinocytosis. We review these recent findings and address how they may be applied to develop new PDAC treatments.

Structural basis of cargo membrane protein discrimination by the human COPII coat machinery.

Genomic analysis shows that the increased complexity of trafficking pathways in mammalian cells involves an expansion of the number of SNARE, Rab and COP proteins. Thus, the human genome encodes four forms of Sec24, the cargo selection subunit of the COPII vesicular coat, and this is proposed to increase the range of cargo accommodated by human COPII‐coated vesicles. In this study, we combined X‐ray crystallographic and biochemical analysis with functional assays of cargo packaging into COPII vesicles to establish molecular mechanisms for cargo discrimination by human Sec24 subunits. A conserved IxM packaging signal binds in a surface groove of Sec24c and Sec24d, but the groove is occluded in the Sec24a and Sec24b subunits. Conversely, LxxLE class transport signals and the DxE signal of VSV glycoprotein are selectively bound by Sec24a and Sec24b subunits. A comparative analysis of crystal structures of the four human Sec24 isoforms establishes the structural determinants for discrimination among these transport signals, and provides a framework to understand how an expansion of coat subunits extends the range of cargo proteins packaged into COPII‐coated vesicles.

Exiting the Endoplasmic Reticulum

Vesicular transport from the endoplasmic reticulum (ER) to the Golgi complex constitutes the initial step in protein secretion. COPII‐coated vesicles mediate the export of newly synthesized proteins from the ER, and this transport step is coupled with COPI‐mediated retrograde traffic to form a transport circuit that supports the compositional asymmetry of the ER–Golgi system. Biochemical and structural studies have advanced our understanding of the mechanisms that control vesicle formation and cargo–protein capture. Recent work has highlighted the function of transitional ER regions in specifying the location of COPII budding.

Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis

NCOA4 is a selective cargo receptor for the autophagic turnover of ferritin, a process critical for regulation of intracellular iron bioavailability. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, we demonstrate that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting. DOI: http://dx.doi.org/10.7554/eLife.10308.001

Mechanisms of Selective Autophagy in Normal Physiology and Cancer

Selective autophagy is critical for regulating cellular homeostasis by mediating lysosomal turnover of a wide variety of substrates including proteins, aggregates, organelles, and pathogens via a growing class of molecules termed selective autophagy receptors. The molecular mechanisms of selective autophagy receptor action and regulation are complex. Selective autophagy receptors link their bound cargo to the autophagosomal membrane by interacting with lipidated ATG8 proteins (LC3/GABARAP) that are intimately associated with the autophagosome membrane. The cargo signals that selective autophagy receptors recognize are diverse but their recognition can be broadly grouped into two classes, ubiquitin-dependent cargo recognition versus ubiquitin-independent. The roles of post-translational modification of selective autophagy receptors in regulating these pathways in response to stimuli are an active area of research. Here we will review recent advances in the identification of selective autophagy receptors and their regulatory mechanisms. Given its importance in maintaining cellular homeostasis, disruption of autophagy can lead to disease including neurodegeneration and cancer. The role of autophagy in cancer is complex as autophagy can mediate promotion or inhibition of tumorigenesis. Here we will also review the importance of autophagy in cancer with a specific focus on the role of selective autophagy receptors.

Stereotactic Body Radiotherapy (SBRT) for Intrahepatic and Hilar Cholangiocarcinoma

Background: Unresectable intrahepatic and hilar cholangiocarcinomas carry a dismal prognosis. Systemic chemotherapy and conventional external beam radiation and brachytherapy have been used with limited success. We explored the use of stereotactic body radiotherapy (SBRT) for these patients. Methods: Patients with unresectable intrahepatic or hilar cholangiocarcinoma or those with positive margins were included in this study. Systemic therapy was used at the discretion of the medical oncologist. The CyberknifeTM stereotactic body radiotherapy system used to treat these patients. Patients were treated with three daily fractions. Clinical and radiological follow-up were performed every three months. Results: 34 patients (16 male and 18 female) with 42 lesions were included in this study. There were 32 unresectable tumors and two patients with resected tumors with positive margins. The median SBRT dose was 30Gy in three fractions. The median follow-up was 38 months (range 8-71 months). The actuarial local control rate was 79%. The median overall survival was 17 months and the median progression free survival was ten months. There were four Grade III toxicities (12%), including duodenal ulceration, cholangitis and liver abscess. Conclusions: SBRT is an effective and reasonably safe local therapy option for unresectable intrahepatic or hilar cholangiocarcinoma.

Compensatory metabolic networks in pancreatic cancers upon perturbation of glutamine metabolism

Pancreatic ductal adenocarcinoma is a notoriously difficult-to-treat cancer and patients are in need of novel therapies. We have shown previously that these tumours have altered metabolic requirements, making them highly reliant on a number of adaptations including a non-canonical glutamine (Gln) metabolic pathway and that inhibition of downstream components of Gln metabolism leads to a decrease in tumour growth. Here we test whether recently developed inhibitors of glutaminase (GLS), which mediates an early step in Gln metabolism, represent a viable therapeutic strategy. We show that despite marked early effects on in vitro proliferation caused by GLS inhibition, pancreatic cancer cells have adaptive metabolic networks that sustain proliferation in vitro and in vivo. We use an integrated metabolomic and proteomic platform to understand this adaptive response and thereby design rational combinatorial approaches. We demonstrate that pancreatic cancer metabolism is adaptive and that targeting Gln metabolism in combination with these adaptive responses may yield clinical benefits for patients.

Stereotactic Body Radiotherapy (SBRT) Reirradiation for Recurrent Pancreas Cancer

Objectives: After adjuvant or definitive radiation for pancreas cancer, there are limited conventional treatment options for recurrent pancreas cancer. We explored the role of (Stereotactic Body Radiotherapy) SBRT for reirradiation of recurrent pancreas Cancer. Methods: This is a retrospective study of patients reirradiated with SBRT for recurrent pancreas cancer. All patients were deemed unresectable and treated with systemic therapy. Fiducial gold markers were used. CT simulation was performed with oral and IV contrast and patients were treated with respiratory motion tracking in the CyberknifeTM system. Results: 30 patients (17 men and 13 women) with a median age of 67 years were included in the study. The median target volume was 41.29cc. The median prescription dose was 25Gy (24-36Gy) in a median of 5 fractions prescribed to a mean 78% isodose line. The median overall survival was 14 months. The 1 and 2 year local control was 78%. The worst toxicity included 3/30(10%) Grade III acute toxicity for pain, bleeding and vomiting. There was 2/30 (7%) Grade III long-term bowel obstructions. Conclusions: SBRT can be a useful and tolerable option for patients with recurrent pancreas cancer after prior radiation.