Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1a, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1a in cells arises from the formation of a disulphide bond-identified by mass spectrometry-between the active-site Cys 94 (connected to Cys 99 in the active enzyme) and Cys 131. Competition between substrate thiols and Cys 131 creates a feedback loop where activation of Ero1a is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1a-Cys131Ala or the isoform Ero1b, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.

OPN (osteopontin) is an integrin-binding highly phosphorylated glycoprotein, recognized as a key molecule in a multitude of biological processes such as bone mineralization, cancer metastasis, cell-mediated immune response, inflammation and cell survival. A significant regulation of OPN function is mediated through PTM (post-translational modification). Using a combination of Edman degradation and MS analyses, we have characterized the complete phosphorylation and glycosylation pattern of native human OPN. A total of 36 phosphoresidues have been localized in the sequence of OPN. There are 29 phosphorylations (Ser8, Ser10, Ser11, Ser46, Ser47, Thr50, Ser60, Ser62, Ser65, Ser83, Ser86, Ser89, Ser92, Ser104, Ser110, Ser113, Thr169, Ser179, Ser208, Ser218, Ser238, Ser247, Ser254, Ser259, Ser264, Ser275, Ser287, Ser292 and Ser294) located in the target sequence of MGCK (mammary gland casein kinase) also known as the Golgi kinase (S/T-X-E/S(P)/D). Six phosphorylations (Ser101, Ser107, Ser175, Ser199, Ser212 and Ser251) are located in the target sequence of CKII (casein kinase II) [S-X-X-E/S(P)/D] and a single phosphorylation, Ser203, is not positioned in the motif of either MGCK or CKII. The 36 phosphoresidues represent the maximal degree of modification since variability at many sites was seen. Five threonine residues are O-glycosylated (Thr118, Thr122, Thr127, Thr131 and Thr136) and two potential sites for N-glycosylation (Asn63 and Asn90) are not occupied in human milk OPN. The phosphorylations are arranged in clusters of three to five phosphoresidues and the regions containing the glycosylations and the RGD (Arg-Gly-Asp) integrin-binding sequence are devoid of phosphorylations. Knowledge about the positions and nature of PTMs in OPN will allow a rational experimental design of functional studies aimed at understanding the structural and functional interdependences in diverse biological processes in which OPN is a key molecule.

We demonstrate temperature-controlled encapsulation and release of the enzyme horseradish peroxidase using a preassembled and covalently closed three-dimensional DNA cage structure as a controllable encapsulation device. The utilized cage structure was covalently closed and composed of 12 double-stranded B-DNA helices that constituted the edges of the structure. The double stranded helices were interrupted by short single-stranded thymidine linkers constituting the cage corners except for one, which was composed by four 32 nucleotide long stretches of DNA with a sequence that allowed them to fold into hairpin structures. As demonstrated by gel-electrophoretic and fluorophore-quenching experiments this design imposed a temperature-controlled conformational transition capability to the structure, which allowed entrance or release of an enzyme cargo at 37 °C while ensuring retainment of the cargo in the central cavity of the cage at 4 °C. The entrapped enzyme was catalytically active inside the DNA cage and was able to convert substrate molecules penetrating the apertures in the DNA lattice that surrounded the central cavity of the cage.

Osteopontin (OPN) is a highly modified integrin-binding protein found in all body fluids. Expression of OPN is strongly correlated with poor prognosis in many different human cancers, suggesting an important but poorly understood role for this protein in tumorigenesis and metastasis. The protein exists in a number of different isoforms differing in the degree of post-translational modifications that are likely to exhibit different functional properties. This study examines for the first time the post-translational modifications of OPN from transformed cells and the effects of these modifications on cell biology. We have characterized the complete phosphorylation and glycosylation patterns of OPN expressed by murine ras-transformed fibroblasts (FbOPN) and differentiating osteoblasts (ObOPN) by a combination of mass spectrometric analyses and Edman degradation. Mass spectrometric analysis showed masses of 34.9 and 35.9 kDa for FbOPN and ObOPN, respectively. Enzymatic dephosphorylation, sequence, and mass analyses demonstrated that FbOPN contains approximately four phosphate groups distributed over 16 potential phosphorylation sites, whereas ObOPN contains ϳ21 phosphate groups distributed over 27 sites. Five residues are O-glycosylated in both isoforms. These residues are fully modified in FbOPN, whereas one site is partially glycosylated in ObOPN. Although both forms of OPN mediated robust integrin-mediated adhesion of mouse ras-transformed fibroblasts, the less phosphorylated FbOPN mediated binding of MDA-MD-435 human tumor cells almost 6-fold more than the heavy phosphorylated ObOPN. These results strongly support the hypothesis that the degree of phosphorylation of OPN produced by different cell types can regulate its function. Osteopontin (OPN)3 is a highly phosphorylated glycoprotein comprising ϳ300 amino acid residues. The protein was first purified from the mineralized matrix of bovine bone (1). However, the presence of OPN is not limited to mineralized tissues but extends to a variety of tissues, cell types, and physiological fluids, including blood, urine, and milk (2). The amino acid sequence of OPN is rich in acidic amino acids and contains an integrin-binding Arg-Gly-Asp (RGD) sequence. OPN is a pleiotropic protein involved in a variety of cellular processes such as migration, adhesion, and signaling (2, 3). OPN is a key molecule in bone remodeling and functions as an inhibitor of ectopic calcification by inhibiting the formation of hydroxylapatite and calcium oxalate (4 -6). Furthermore, OPN is implicated in diverse biological processes, including tumorigenesis, metastasis, cytokine production, wound healing, autoimmune disease, and stroke (3, 7-10). OPN has recently been demonstrated to be required for mucosal protection in acute inflammatory colitis (11).OPN-integrin interaction controls many aspects of cell behavior, including cell attachment, migration, chemotaxis, and immune modulation in various cell types (2, 12). The ␣ v ␤ 6 , ␣ 5 ␤ 1 , ␣ 8 ␤ 1 , ␣ v ␤ 1 , ␣ v ␤ 5 , and ␣ v ␤ 3 integrins recog...

Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. Osteopontin (OPN) 2 is a highly acidic phosphorylated glycoprotein containing an integrin-binding Arg-Gly-Asp (RGD) sequence. OPN is implicated in a diversity of physiological processes such as bone mineralization, inhibition of ectopic calcification, wound healing, inflammation, regulation of immune cell functions, and tumor growth (1-3). OPN is synthesized by a variety of cells and is present in most tissues and body fluids, including blood, urine, and milk (1).OPN is present in milk in very high concentrations (ϳ138 mg/liter) (4), but the function is not clear. However, several functions can be hypothesized, for instance OPN can inhibit the formation of renal stones by inhibiting growth and aggregation of calcium crystals (1). Similarly, OPN can be speculated to inhibit unintentional calcium crystallization and precipitation in milk. OPN has also been implicated in mammary gland development and differentiation (5). Furthermore, a significant proportion of the milk OPN is expected to pass through the gut and into the intestines largely intact upon milk consumption, as the protein is relatively resistant to proteolysis by neonatal gastric juice (6). This opens the possibility that OPN or OPN fragments could play a role in the infant immune response, as milk OPN can induce the expression of interleukin-12 from human intestinal lamina propria mononuclear cells (4).Many of the cellular functions propagated by OPN are mediated through interactions with integrin receptors. The ␣ v ␤ 6 ,, and ␣ v ␤ 3 integrins bind OPN via the RGD sequence (1,7,8), whereas the ␣ 4 ␤ 1 and ␣ 9 ␤ 1 integrins bind the cryptic non-RGD motif Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) (9, 10), and the monocyte ␣ X ␤ 2 -integrin receptor interacts with the highly acidic parts of OPN (11).OPN is extensively altered through post-translational modifications such as phosphorylation, sulfation, and glycosylation. These modifications have significant implications on the interaction with integrins (8). Another modification that can alter the functionality of OPN is proteolytic processing. Recently, intracellular cleavage of OPN by caspase-8 at Asp 119 -Phe 120 and Asp 141 -Gly 142 has been shown to be a regulatory switch in determining cell death of cancer cells (12). OPN is also a substrate for thrombin and matrix metalloproteinase (MMP)-2, -3, -7, and -9 (13-17). Thrombin hydrolyzes human OPN at Arg 152 -Ser 153 , whereas MMPs cleave nearby at Gly 150 -Leu 151 , which in all cases results in the generation of N-terminal OPN fragments containing an exposed RGD 145 sequence. Thrombin and MMP cleavage of OPN ...