The Neuropilins (Nrps) are a family of essential cell surface receptors involved in multiple fundamental cellular signaling cascades. Nrp family members have key functions in VEGF-dependent angiogenesis and semaphorin-dependent axon guidance, controlling signaling and cross-talk between these fundamental physiological processes. More recently, Nrp function has been found in diverse signaling and adhesive functions, emphasizing their role as pleiotropic co-receptors. Pathological Nrp function has been shown to be important in aberrant activation of both canonical and alternative pathways. Here we review key recent insights into Nrp function in human health and disease.The Nrps 2 are essential cell surface receptors with pleiotropic function in human health, functioning in many key biological processes including in the cardiovascular, neuronal, and immune systems (1). The two Nrp family members, Nrp1 and Nrp2, are type I transmembrane proteins that are conserved in all vertebrates and are ϳ40% identical at the amino acid level with a conserved domain structure. The Nrp extracellular region possesses five structured domains that are essential for ligand binding, a single transmembrane domain, and a short intracellular domain that possesses a PSD-95/Dlg/ZO-1 (PDZ)-binding motif (1).

Plants use the insoluble polyglucan starch as their primary glucose storage molecule. Reversible phosphorylation, at the C6 and C3 positions of glucose moieties, is the only known natural modification of starch and is the key regulatory mechanism controlling its diurnal breakdown in plant leaves. The glucan phosphatase Starch Excess4 (SEX4) is a position-specific starch phosphatase that is essential for reversible starch phosphorylation; its absence leads to a dramatic accumulation of starch in Arabidopsis, but the basis for its function is unknown. Here we describe the crystal structure of SEX4 bound to maltoheptaose and phosphate to a resolution of 1.65 Å. SEX4 binds maltoheptaose via a continuous binding pocket and active site that spans both the carbohydrate-binding module (CBM) and the dual-specificity phosphatase (DSP) domain. This extended interface is composed of aromatic and hydrophilic residues that form a specific glucan-interacting platform. SEX4 contains a uniquely adapted DSP active site that accommodates a glucan polymer and is responsible for positioning maltoheptaose in a C6-specific orientation. We identified two DSP domain residues that are responsible for SEX4 sitespecific activity and, using these insights, we engineered a SEX4 double mutant that completely reversed specificity from the C6 to the C3 position. Our data demonstrate that the two domains act in consort, with the CBM primarily responsible for engaging glucan chains, whereas the DSP integrates them in the catalytic site for position-specific dephosphorylation. These data provide important insights into the structural basis of glucan phosphatase site-specific activity and open new avenues for their biotechnological utilization.tarch is the primary carbohydrate storage molecule in plants and is an essential constituent of human and animal diets. Starch granules are composed of the glucose homopolymers amylose (10-25%) and amylopectin (75-90%) (1, 2). Amylose is a linear molecule formed from α-1,4-glycosidic-linked chains, whereas amylopectin is formed from α-1,4-glycosidic-linked chains with α-1,6-glycosidic-linked branches (3, 4). Adjacent amylopectin chains interact to form double helices that cause starch granules to be water insoluble, which is an essential feature for its function as a glucose storage molecule (1, 3, 5). However, the outer granular surface of transitory starch must be solubilized during nonphotosynthetic periods so that glycolytic enzymes can access and degrade starch glucans and meet the metabolic needs of the plant (6, 7). Plants regulate the solubility of the starch granular surface via reversible starch phosphorylation that results in a cyclic degradative process: phosphorylation by dikinases, degradation by starch hydrolyzing amylases, and dephosphorylation by phosphatases (1, 8-11). Phosphorylation of amylopectin chains causes helical unwinding and local solubilization of the outer starch granule (12-14). The local solubilization and helix unwinding permits degradation of surface, linear α-1,4 glucan chai...

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet. 10, e1004465). However, the extent of decrease appears to be tissue-and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation.Collagens comprise a large family of structurally related extracellular matrix proteins (1). Among all of the genetic types of collagen identified, fibrillar type I collagen is the most abundant, providing tissues and organs with form and stability. It is a heterotrimeric molecule composed of two ␣1 chains and one ␣2 chain forming a long uninterrupted triple helix with short non-helical domains (telopeptide) at both N and C termini. One of the functionally important characteristics of collagen is its unique, sequential post-translational modifications of Lys residues. The modifications include hydroxylation and monoand diglycosylation of hydroxylysine (Hyl), 3 and oxidative deamination of Lys and Hyl in the N-and C-telopeptides followed by extensive covalent intermolecular cross-linking (2). It is now clear that defective Lys modifications of collagen cause and/or are associated with a broad range of connective tissue disorders, including Ehlers-Danlos syndrome type VIA (3), bronchopulmonary dysplasia (4), Kuskokwim syndrome (5), Bruck syndrome (6, 7), fibrosis (8), disuse osteoporosis (9), and cancer progression (10, 11). Our recent finding showed that a switch of collage...

Starch is a water-insoluble, Glc-based biopolymer that is used for energy storage and is synthesized and degraded in a diurnal manner in plant leaves. Reversible phosphorylation is the only known natural starch modification and is required for starch degradation in planta. Critical to starch energy release is the activity of glucan phosphatases; however, the structural basis of dephosphorylation by glucan phosphatases is unknown. Here, we describe the structure of the Arabidopsis thaliana starch glucan phosphatase LIKE SEX FOUR2 (LSF2) both with and without phospho-glucan product bound at 2.3Å and 1.65Å, respectively. LSF2 binds maltohexaose-phosphate using an aromatic channel within an extended phosphatase active site and positions maltohexaose in a C3-specific orientation, which we show is critical for the specific glucan phosphatase activity of LSF2 toward native Arabidopsis starch. However, unlike other starch binding enzymes, LSF2 does not possess a carbohydrate binding module domain. Instead we identify two additional glucan binding sites located within the core LSF2 phosphatase domain. This structure is the first of a glucan-bound glucan phosphatase and provides new insights into the molecular basis of this agriculturally and industrially relevant enzyme family as well as the unique mechanism of LSF2 catalysis, substrate specificity, and interaction with starch granules.

No abstract