Search

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.

BACKGROUND & AIMS: Contrast enhanced computed tomography (CT-scan) is a standard of care for the radiological diagnosis of hepatocellular carcinoma (HCC) in patients with cirrhosis. This technique, however, is not validated to exclude intrahepatic cholangiocarcinoma (ICC) which may develop in patients with cirrhosis, as well. METHODS: To assess the features of contrast CT-scan in the diagnosis of ICC, we reviewed all CT-scan films obtained in cirrhotic patients with a histologically documented ICC, taking in consideration the pattern and dynamics of the arterial, portal venous and delayed phases of contrast uptake. RESULTS: Thirty-two patients had 40 nodules of ICC (22 male; median age 62years; 13 hepatitis C) that were identified either during surveillance with abdominal ultrasound (21 patients, 66%) or incidentally (11 patients, 34%). ICC was either multifocal or ≥ 30 mm in 11 of the former and 10 of the latter group (52% vs. 91%, p

AIM: Primary aim was to validate the percentage of intrahepatic cholangiocarcinomas (ICC) which have a contrast vascular pattern at contrast enhanced ultrasound (CEUS) at risk of misdiagnosis with hepatocellular carcinoma (HCC) and, secondary aim, to verify if any characteristics in the CEUS pattern helps to identify ICC. METHODS: All ICC on cirrhosis seen in three Italian centres (Bologna, Milan and Pavia) between 2003 and 2011, in which CEUS and at least another imaging technique (CT or MRI) had been performed, were retrospectively identified. Those patients with ICC size comparable to the early HCC stage (Milan criteria, considered as small ICC) were enrolled for this study. The enhancement pattern at CEUS was analysed and compared with CT or MRI. RESULTS: A total of 25 small ICC made this study group. CEUS was at risk of misdiagnosis of ICC for HCC in a significantly higher number of cases than in CT (performed in 24 ICC) (52% vs. 4.2%, P = 0.009) and MRI (11 ICC) (52% vs. 9.1%, P = 0.02). A different contrast pattern among all techniques was found in 6 of 10 ICC lesions submitted to the three imaging methods. In the arterial phase, ICC lacked global hyperenhacement in approximately 50% of cases at CEUS and the degree of intensity of wash-out in the late phase was marked in 24% of nodules. CONCLUSIONS: CEUS misdiagnosed as HCC a significantly higher number of ICC lesions in cirrhotic patients than CT and MRI. However, some CEUS contrast features can help suspect ICC, especially in some cases with inconclusive CT or MRI.

ERp44 mediates thiol-dependent retention in the early secretory pathway, forming mixed disulphides with substrate proteins through its conserved CRFS motif. Here, we present its crystal structure at a resolution of 2.6 A. Three thioredoxin domains—a, b and b′—are arranged in a clover-like structure. A flexible carboxy-terminal tail turns back to the b′ and a domains, shielding a hydrophobic pocket in domain b′ and a hydrophobic patch around the CRFS motif in domain a. Mutational and functional studies indicate that the C-terminal tail gates the CRFS area and the adjacent hydrophobic pocket, dynamically regulating protein quality control.

Medullary cystic kidney disease/familial juvenile hyperuricemic nephropathy (MCKD/FJHN) are autosomal dominant renal disorders characterized by tubulo-interstitial fibrosis, hyperuricemia and medullary cysts. They are caused by mutations in the gene encoding uromodulin, the most abundant protein in urine. Uromodulin (or Tamm–Horsfall protein) is a glycoprotein that is exclusively expressed by epithelial tubular cells of the thick ascending limb of Henle’s loop and distal convoluted tubule. To date, 37 different uromodulin mutations have been described in patients with MCKD/FJHN. Interestingly, 60% of them involve one of the 48 conserved cysteine residues. We have previously shown that cysteine-affecting mutations could lead to partial endoplasmic reticulum (ER) retention. In this study, as a further step in understanding uromodulin biology in health and disease, we provide the first extensive study of intracellular trafficking and subcellular localization of wild-type and mutant uromodulin isoforms. We analyzed a set of 12 different uromodulin mutations that were representative of the different kind of mutations identified so far by different experimental approaches (immunofluorescence, electron microscopy, biochemistry and in vivo imaging) in transiently transfected HEK293 and Madin–Darby canine kidney cells. We assessed protein processing in the secretory pathway and could demonstrate that although to different extent, all uromodulin mutations lead to defective ER to Golgi protein transport, suggesting a common pathogenetic mechanism in MCKD/FJHN.