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Significance This study provides a structure for microsomal triglyceride transfer protein, a key protein in lipid metabolism and transport. Microsomal triglyceride transfer protein is linked to a human disease state, abetalipoproteinemia. The structure helps us to understand how this protein functions and gives a rationale for how previously reported mutations result in loss of function of the protein and hence, cause disease. The structure also provides a means for rational drug design to treat cardiovascular disease, hypercholesterolemia, and obesity. Microsomal triglyceride transfer protein is composed of 2 subunits. The β-subunit, protein disulfide isomerase (PDI), also acts independently as a protein folding catalyst. The structure that we present here gives insights into how PDI functions in protein folding., Microsomal triglyceride transfer protein (MTP) plays an essential role in lipid metabolism, especially in the biogenesis of very low-density lipoproteins and chylomicrons via the transfer of neutral lipids and the assembly of apoB-containing lipoproteins. Our understanding of the molecular mechanisms of MTP has been hindered by a lack of structural information of this heterodimeric complex comprising an MTPα subunit and a protein disulfide isomerase (PDI) β-subunit. The structure of MTP presented here gives important insights into the potential mechanisms of action of this essential lipid transfer molecule, structure-based rationale for previously reported disease-causing mutations, and a means for rational drug design against cardiovascular disease and obesity. In contrast to the previously reported structure of lipovitellin, which has a funnel-like lipid-binding cavity, the lipid-binding site is encompassed in a β-sandwich formed by 2 β-sheets from the C-terminal domain of MTPα. The lipid-binding cavity of MTPα is large enough to accommodate a single lipid. PDI independently has a major role in oxidative protein folding in the endoplasmic reticulum. Comparison of the mechanism of MTPα binding by PDI with previously published structures gives insights into large protein substrate binding by PDI and suggests that the previous structures of human PDI represent the “substrate-bound” and “free” states rather than differences arising from redox state.

The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility. As an important factor for proper IgM maturation and hence immune function, pERp1 is upregulated in many auto-immune diseases. This makes it a potential therapeutic target. pERp1 belongs to the CNPY family of ER resident saposin-like proteins. To date, five of these proteins have been identified. All are implicated in protein folding and all contain a saposin-like domain. All previously structurally characterized saposins are involved in lipid binding. However, there are no reports of CNPY family members interacting with lipids, suggesting a novel function for the saposin fold. However, the molecular mechanisms of their function remain elusive. To date, no structure of any CNPY protein has been reported. Here, we present the high-resolution (1.4 A) crystal structure of human pERp1 and confirm that it has a saposin-fold with unique structural elements not present in other saposin-fold structures. The implications for the role of CNPY proteins in protein folding in the ER are discussed.

The EGFR adaptor protein, CIN85, has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill defined. Here we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase, PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SRC homology 3 domains of CIN85 interacted with the proline-arginine–rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF degradation. This activity is essential in vivo, as specific loss of the CIN85–PHD2 interaction in CRISPR/Cas9-edited cells affected growth and migration properties, as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2 and uncovered an essential survival function in tumor cells by linking growth factor adaptors with hypoxia signaling. Significance: This study provides unprecedented evidence for an oxygen-independent mechanism of PHD2 regulation that has important implications in cancer cell survival.

A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis.

Coronary artery disease is the most common cause of death globally and is linked to a number of risk factors including serum low density lipoprotein, high density lipoprotein, triglycerides and lipoprotein(a). Recently two proteins, angiopoietin-like protein 3 and 4, have emerged from genetic studies as being factors that significantly modulate plasma triglyceride levels and coronary artery disease. The exact function and mechanism of action of both proteins remains to be elucidated, however, mutations in these proteins results in up to 34% reduction in coronary artery disease and inhibition of function results in reduced plasma triglyceride levels. Here we report the crystal structures of the fibrinogen-like domains of both proteins. These structures offer new insights into the reported loss of function mutations, the mechanisms of action of the proteins and open up the possibility for the rational design of low molecular weight inhibitors for intervention in coronary artery disease., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe; 1048

Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl(2) (2.1 A; R = 18.2%, R(free) = 21.9%), and in complex with glutamate, MgCl(2), and ADP (2.7 A; R = 19.0%, R(free) = 24.2%). Inspection of these structures reveals an unusual binding pocket for the alpha-carboxylate of the glutamate substrate and an ATP-independent Mg(2+) coordination site, clarifying the Mg(2+) dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.

P58(IPK) is one of the endoplasmic reticulum- (ER-) localised DnaJ (ERdj) proteins which interact with the chaperone BiP, the mammalian ER ortholog of Hsp70, and are thought to contribute to the specificity and regulation of its diverse functions. P58(IPK), expression of which is upregulated in response to ER stress, has been suggested to act as a co-chaperone, binding un- or misfolded proteins and delivering them to BiP. In order to give further insights into the functions of P58(IPK), and the regulation of BiP by ERdj proteins, we have determined the crystal structure of human P58(IPK) to 3.0 A resolution using a combination of molecular replacement and single wavelength anomalous diffraction. The structure shows the human P58(IPK) monomer to have a very elongated overall shape. In addition to the conserved J domain, P58(IPK) contains nine N-terminal tetratricopeptide repeat motifs, divided into three subdomains of three motifs each. The J domain is attached to the C-terminal end via a flexible linker, and the structure shows the conserved Hsp70-binding histidine-proline-aspartate (HPD) motif to be situated on the very edge of the elongated protein, 100 A from the putative binding site for unfolded protein substrates. The residues that comprise the surface surrounding the HPD motif are highly conserved in P58(IPK) from other organisms but more varied between the human ERdj proteins, supporting the view that their regulation of different BiP functions is facilitated by differences in BiP-binding.

Glutathione (L-γ-glutamyl-L-cysteinyl-glycine; GSH) is an abundant tripeptide critical for oxidative stress response and detoxification of xenobiotics. It contributes to signalling pathways as well as the regulation of enzymatic activity by altering the accessibility of protein sulfhydryl groups (1, 2). In addition to serving as a redox buffer, glutathione participates in the storage and transport of cysteine (3) and select heavy metals (4). The diverse functions of glutathione are necessary for normal cellular processes (3, 5–7), and disruption of glutathione homeostasis is associated with numerous disease states (8, 9). Glutathione is synthesized in humans by the sequential action of glutamate cysteine ligase (GCL) and glutathione synthetase (10). GCL catalyzes the formation of an amide linkage between the γ-carboxyl group of glutamate and cysteine to form γ-glutamylcysteine, which is the rate-limiting step in glutathione biosynthesis. In vertebrate systems, GCL typically exists as a heterodimer consisting of a catalytic subunit (73 kDa) and a modifier subunit (31 kDa). The catalytic subunit (GCLC) contains the active site while the modifier subunit (GCLM) participates in regulation of enzymatic activity, enhancing catalytic efficiency upon heterodimer formation (11). GCLC null mice are embryonic lethal (7, 12) while GCLM null mice are viable, producing low levels of glutathione (10%–20% of normal levels), consistent with a regulatory role (6). Several reports of hereditary GCL deficiency in humans have been communicated (13–17). The disease is marked by hemolytic anemia, low levels of erythrocyte glutathione (typically < 10% of normal levels), and in some cases, neurological disability. In the past decade, specific mutations in the coding region of GCLC have been identified in patients with hereditary GCL deficiency. Of the four clinical mutants identified thus far, three result in a leucine substitution: proline 158 (Pro158Leu), histidine 370 (His370Leu), or proline 414 (Pro414Leu). In each case, GCL activity was markedly reduced in erythrocyte samples with corresponding reductions in glutathione levels. However, the precise mechanism by which the mutation reduced enzymatic activity was not identified beyond speculation that each mutation may impact protein stability. A fourth mutation, resulting in the substitution of an arginine residue at position 127 with a cysteine residue (Arg127Cys), has been shown to directly impair enzymatic activity (14). Recently, the crystal structure of the closely related Saccharomyces cerevisiae GCL was reported, allowing a homology model of human GCLC to be generated (Figure 1) (18). Examination of the model provided significant insights to the possible functions of each of these four residues. In the current study, we used GCLC null mouse embryonic fibroblasts (7) and S. cerevisiae devoid of glutamate cysteine ligase (Δgsh1) as model systems to further examine the impacts of these clinical mutations on glutathione production. In addition, we have kinetically characterized each of the human GCLC clinical mutants, either alone or in complex with human GCLM. The results of these studies demonstrate the impact of the clinical mutations responsible for hereditary GCL deficiency on the activity and stability of the enzyme in vitro and in vivo, as well as the critical role of the modifier subunit in enhancing wild-type activity and restoring mutant activity. Figure 1 Homology model of human GCLC reveals the locations of the four clinical mutations