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Summary To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44’s active cysteine, simultaneously unmask the substrate binding site and −RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles., Graphical Abstract, Highlights • ERp44 governs a pH-regulated assembly control cycle in the early secretory pathway • Accessibility of ERp44’s active site and –RDEL ER retrieval motif is pH dependent • Unmasking of ERp44’s active site likely involves protonation of cysteine 29 • ERp44 captures client proteins at cisGolgi-equivalent pH for retrieval to the ER

A multicenter randomized controlled trial established sorafenib as a standard of care for patients with advanced hepatocellular carcinoma (HCC). Because the study was prematurely interrupted due to survival benefits in the sorafenib arm, we conducted an observational study to adequately assess risks and benefits of this regimen in field practice. Starting in 2008, all clinically compensated patients with advanced HCC and those with an intermediate HCC who were unfit or failed to respond to ablative therapies were consecutively evaluated in six liver centers in Italy, for tolerability as well as radiologic and survival response to 800-mg/d sorafenib therapy. Treatment was down-dosed or interrupted according to drug label. Two hundred ninety-six patients (88% Child-Pugh A, 75% Barcelona Clinic Liver Cancer [BCLC]-C, and 25% BCLC-B) received sorafenib for 3.8 months (95% CI 3.3-4.4). Two hundred sixty-nine (91%) patients experienced at least one adverse event (AE), whereas 161 (54%) had to reduce dosing. Treatment was interrupted in 103 (44%) for disease progression, in 95 (40%) for an AE, and in 38 (16%) for liver deterioration. The median survival was 10.5 months in the overall cohort, 8.4 months in BCLC-C versus 20.6 months in BCLC-B patients (P < 0.0001), and 21.6 months in the 77 patients treated for >70% of the time with a half dose versus 9.6 months in the 219 patients treated for >70% of the time with a full dose. At month 2 of treatment, the overall radiologic response was 8%. Eastern Cooperative Oncology Group performance status, macrovascular invasion, extrahepatic spread of the tumor, radiologic response at month 2, and sorafenib dosing were independent predictors of shortened survival. Conclusion: Overall, safety, effectiveness, and generalizability of sorafenib therapy in HCC was validated in field practice. The effectiveness of half-dosed sorafenib may have implications for tailored therapy.

BACKGROUND AND AIMS: Although contrast-enhanced computed tomography (CT), dynamic magnetic resonance (MRI) and fine needle biopsy (FNB) are the standard of care to diagnose hepatocellular carcinoma (HCC), the clinical and economic benefits of the updated AASLD diagnostic algorithm, including the drop of contrast enhanced ultrasound (CEUS), have not been previously evaluated. METHODS: 119 de novo liver nodules detected during ultrasound (US) surveillance in 98 cirrhotics, 7 2cm in size, were sequentially examined by CEUS and CT, using MRI as a rescue approach in patients lacking a typical vascular pattern for HCC by one or both contrast techniques in the 1-2cm nodules and by CT in the >2cm nodules. A FNB was performed when required to meet both 2005 and 2010 AASLD criteria. RESULTS: Eighty-four (70%) nodules were HCC: the radiological diagnosis was done in 38 (88%) of those 1-2cm and in 38 (95%) for those >2cm HCCs according to 2010 AASLD criteria. CT or MRI detected 13 HCC nodules that were missed by unenhanced US. Despite an absolute specificity, CEUS failed to identify any HCC uncharacterized by CT or MRI. By updated AASLD criteria, 6 (17%) FNB procedures were spared in patients with 1-2cm nodules (p=0.025), as compared to 2005 criteria. The 2010 vs. 2005 AASLD per patient cost was similar in 1-2cm nodules, 432 € vs. 451 € (p=0.46), but lower in >2cm nodules, 248 € vs. 321 € (p

In the early secretory compartment(ESC), a network of chaperones and enzymes assist oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin-4 (Prx4) canutilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC bearing KDEL-like motifs. PDI binds preferentially Ero1α, while ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.

In the endoplasmic reticulum (ER) of human cells, ERO1a and protein-disulfide isomerase (PDI) constitute one of the major electron flow pathways that catalyze oxidative folding of secretory proteins. Specific and limited PDI oxidation by ERO1a is essential to avoid ER hyperoxidation. To investigate how ERO1a oxidizes PDI selectively among more than 20 ER resident PDI family member proteins, we performed docking simulations and systematic biochemical analyses. Our findings reveal that a protruding b-hairpin of ERO1a specifically interacts with the hydrophobic pocket present in the redox-inactive PDI b"-domain through the stacks between their aromatic residues, leading to preferred oxidation of the C-terminal PDI a"-domain. ERO1a associated preferentially with reduced PDI, explaining the stepwise disulfide shuttle mechanism, first from ERO1a to PDI and then from oxidized PDI to an unfolded polypeptide bound to its hydrophobic pocket. The interaction of ERO1! with ERp44, another PDI family member protein, was also analyzed. Notably, ERO1a-dependent PDI oxidation was inhibited by a hyperactive ERp44 mutant that lacks the C-terminal tail concealing the substrate-binding hydrophobic regions. The potential ability of ERp44 to inhibit ERO1a activity may suggest its physiological role in ER redox and protein homeostasis.

The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in theendoplasmic reticulum (ER) and in storage vacuoles, respectively.We show here that a chimeric protein composed of phaseolinand 89 amino acids of g-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-typeg-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin,the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). Arelevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chaselabeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that alsoextensively interact with BiP, zeolin is highly stable. We conclude that the g-zein portion is sufficient to induce the formation ofprotein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionallycomplement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimericstorage proteins.

The binding protein (BiP; a member of the heat-shock 70 family) is a major chaperone of the endoplasmic reticulum (ER). Interactions with BiP are believed to inhibit unproductive aggregation of newly synthesized secretory proteins during folding and assembly. In vitro, BiP has a preference for peptide sequences enriched in hydrophobic amino acids, which are expected to be exposed only in folding and assembly intermediates or in defective proteins. However, direct information regarding sequences recognized in vivo by BiP on real proteins is very limited. We have shown previously that newly synthesized monomers of the homotrimeric storage protein phaseolin associate with BiP and that phaseolin trimerization in the ER abolishes such interactions. Using different phaseolin constructs and green fluorescent protein (GFP) fusion proteins, we show here that one of the two alpha-helical regions of polypeptide contact in phaseolin trimers (35 amino acids located close to the C terminus and containing three potential BiP binding sites) effectively promotes BiP association with phaseolin and with secretory GFP fusions expressed in transgenic tobacco or in transfected protoplasts. We also show that overexpressed BiP transiently sequesters phaseolin polypeptides. We conclude that one of the regions of monomer contact is a BiP binding determinant and suggest that during the synthesis of phaseolin, the association with BiP and trimer formation are competing events. Finally, we show that the other, internal region of contact between monomers is necessary for phaseolin assembly in vivo and contains one potential BiP binding site.