SDS22 selectively recognizes and traps metal-deficient inactive PP1.

The metalloenzyme protein phosphatase 1 (PP1), which is responsible for ≥50% of all dephosphorylation reactions, is regulated by scores of regulatory proteins, including the highly conserved SDS22 protein. SDS22 has numerous diverse functions, surprisingly acting as both a PP1 inhibitor and as an activator. Here, we integrate cellular, biophysical, and crystallographic studies to address this conundrum. We discovered that SDS22 selectively binds a unique conformation of PP1 that contains a single metal (M2) at its active site, i.e., SDS22 traps metal-deficient inactive PP1. Furthermore, we showed that SDS22 dissociation is accompanied by a second metal (M1) being loaded into PP1, as free metal cannot dissociate the complex and M1-deficient mutants remain constitutively trapped by SDS22. Together, our findings reveal that M1 metal loading and loss are essential for PP1 regulation in cells, which has broad implications for PP1 maturation, activity, and holoenzyme subunit exchange.

Structure and evolution of ENTH and VHS/ENTH-like domains in tepsin.

Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein 4 (AP4)-coated vesicles originating at the trans-Golgi network (TGN). The molecular basis for interactions between AP4 subunits and motifs in the tepsin C-terminus have been characterized, but the biological role of tepsin remains unknown. We determined X-ray crystal structures of the tepsin epsin N-terminal homology (ENTH) and VHS/ENTH-like domains. Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins. The tepsin ENTH domain lacks helix0, helix8 and a lipid binding pocket found in epsin1/2/3. These results explain why tepsin requires AP4 for its membrane recruitment and further suggest ENTH domains cannot be defined solely as lipid binding modules. The VHS domain lacks helix8 and thus contains fewer helices than other VHS domains. Structural data explain biochemical and biophysical evidence that tepsin VHS does not mediate known VHS functions, including recognition of dileucine-based cargo motifs or ubiquitin. Structural comparisons indicate the domains are very similar to each other, and phylogenetic analysis reveals their evolutionary pattern within the domain superfamily. Phylogenetics and comparative genomics further show tepsin within a monophyletic clade that diverged away from epsins early in evolutionary history (~1500 million years ago). Together, these data provide the first detailed molecular view of tepsin and suggest tepsin structure and function diverged away from other epsins. More broadly, these data highlight the challenges inherent in classifying and understanding protein function based only on sequence and structure.

A gatekeeper chaperone complex directs translocator secretion during type three secretion.

Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ∼20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.

The Chlamydia effector chlamydial outer protein N (CopN) sequesters tubulin and prevents microtubule assembly.

Chlamydia species are obligate intracellular pathogens that utilize a type three secretion system to manipulate host cell processes. Genetic manipulations are currently not possible in Chlamydia, necessitating study of effector proteins in heterologous expression systems and severely complicating efforts to relate molecular strategies used by Chlamydia to the biochemical activities of effector proteins. CopN is a chlamydial type three secretion effector that is essential for virulence. Heterologous expression of CopN in cells results in loss of microtubule spindles and metaphase plate formation and causes mitotic arrest. CopN is a multidomain protein with similarity to type three secretion system "plug" proteins from other organisms but has functionally diverged such that it also functions as an effector protein. We show that CopN binds directly to αß-tubulin but not to microtubules (MTs). Furthermore, CopN inhibits tubulin polymerization by sequestering free αß-tubulin, similar to one of the mechanisms utilized by stathmin. Although CopN and stathmin share no detectable sequence identity, both influence MT formation by sequestration of αß-tubulin. CopN displaces stathmin from preformed stathmin-tubulin complexes, indicating that the proteins bind overlapping sites on tubulin. CopN is the first bacterial effector shown to disrupt MT formation directly. This recognition affords a mechanistic understanding of a strategy Chlamydia species use to manipulate the host cell cycle.

Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase.

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.

The structure of SDS22 provides insights into the mechanism of heterodimer formation with PP1.

Protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets by associating with nearly 200 regulatory proteins to form highly specific holoenzymes. The vast majority of regulators are intrinsically disordered proteins (IDPs) and bind PP1 via short linear motifs within their intrinsically disordered regions. One of the most ancient PP1 regulators is SDS22, a protein that is conserved from yeast to mammals. Sequence analysis of SDS22 revealed that it is a leucine-rich repeat (LRR) protein, suggesting that SDS22, unlike nearly every other known PP1 regulator, is not an IDP but instead is fully structured. Here, the 2.9 Šresolution crystal structure of human SDS22 in space group P212121 is reported. SDS22 adopts an LRR fold with the horseshoe-like curvature typical for this family of proteins. The structure results in surfaces with distinct chemical characteristics that are likely to be critical for PP1 binding.

AP-4 vesicles contribute to spatial control of autophagy via RUSC-dependent peripheral delivery of ATG9A.

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including 'Dynamic Organellar Maps', to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the trans-Golgi network to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the "ATG9A reservoir" required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

Oxidant stress-induced loss of IRS-1 and IRS-2 proteins in rat skeletal muscle: role of p38 MAPK.

Oxidative stress is characterized as an imbalance between the cellular production of oxidants and the cellular antioxidant defenses and contributes to the development of numerous cardiovascular and metabolic disorders, including hypertension and insulin resistance. The effects of prolonged oxidant stress in vitro on the insulin-dependent glucose transport system in mammalian skeletal muscle are not well understood. This study examined the in vitro effects of low-level oxidant stress (60-90 microM, H(2)O(2)) for 4 h on insulin-stimulated (5 mU/ml) glucose transport activity (2-deoxyglucose uptake) and on protein expression of critical insulin signaling factors (insulin receptor (IR), IR substrates IRS-1 and IRS-2, phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3)) in isolated soleus muscle of lean Zucker rats. This oxidant stress exposure caused significant (50%, p<0.05) decreases in insulin-stimulated glucose transport activity that were associated with selective loss of IRS-1 (59%) and IRS-2 (33%) proteins, increased (64%) relative IRS-1 Ser(307) phosphorylation, and decreased phosphorylation of Akt Ser(473) (50%) and GSK-3beta Ser(9) (43%). Moreover, enhanced (37%) phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was observed. Selective inhibition of p38 MAPK (10 microM A304000) prevented a significant portion (29%) of the oxidant stress-induced loss of IRS-1 (but not IRS-2) protein and allowed partial recovery of the impaired insulin-stimulated glucose transport activity. These results indicate that in vitro oxidative stress in mammalian skeletal muscle leads to substantial insulin resistance of distal insulin signaling and glucose transport activity, associated with a selective loss of IRS-1 protein, in part due to a p38 MAPK-dependent mechanism.

Interactions of the advanced glycation end product inhibitor pyridoxamine and the antioxidant alpha-lipoic acid on insulin resistance in the obese Zucker rat.

Oxidative stress and protein glycation can contribute to the development of insulin resistance and complications associated with type 2 diabetes mellitus. The antioxidant alpha-lipoic acid (ALA) reduces oxidative stress and the formation of advanced glycation end products (AGEs) and improves insulin sensitivity in skeletal muscle and liver. The AGE inhibitor pyridoxamine (PM) prevents irreversible protein glycation, thereby reducing various diabetic complications. The potential interactive effects of ALA and PM in the treatment of whole-body and skeletal muscle insulin resistance have not been investigated. Therefore, this study was designed to determine the effects of combined ALA and PM treatments on reducing muscle oxidative stress and ameliorating insulin resistance in prediabetic obese Zucker rats. Obese Zucker rats were assigned to either a control group or to a treatment group receiving daily injections of the R-(+)-enantiomer of ALA (R-ALA, 92 mg/kg) or PM (60 mg/kg), individually or in combination, for 6 weeks. The individual and combined treatments with R-ALA and PM were effective in significantly (P < .05) reducing plantaris muscle protein carbonyls (33%-40%) and urine-conjugated dienes (22%-38%), markers of oxidative stress. The R-ALA and PM in combination resulted in the largest reductions of fasting plasma glucose (23%), insulin (16%), and free fatty acids (24%) and of muscle triglycerides (45%) compared with alterations elicited by individual treatment with R-ALA or PM. Moreover, the combination of R-ALA and PM elicited the greatest enhancement of whole-body insulin sensitivity both in the fasted state and during an oral glucose tolerance test. Finally, combined R-ALA/PM treatments maintained the 44% enhancement of in vitro insulin-mediated glucose transport activity in soleus muscle of obese Zucker rats treated with R-ALA alone. Collectively, these results document a beneficial interaction of the antioxidant R-ALA and the AGE inhibitor PM in the treatment of whole-body and skeletal muscle insulin resistance in obese Zucker rats.