Glycan-independent role of calnexin in the intracellular retention of Charcot-Marie-tooth 1A Gas3/PMP22 mutants

Missense point mutations in Gas3/PMP22 are responsible for the peripheral neuropathies Charcot-Marie-Tooth 1A and Dejerine Sottas syndrome. These mutations induce protein misfolding with the consequent accumulation of the proteins in the endoplasmic reticulum and the formation of aggresomes. During folding, Gas3/PMP22 associates with the lectin chaperone calnexin. Here, we show that calnexin interacts with the misfolded transmembrane domains of Gas3/PMP22, fused to green fluorescent protein, in a glycan-independent manner. In addition, photobleaching experiments in living cells revealed that Gas3/PMP22-green fluorescent protein mutants are mobile but diffuse at almost half the diffusion coefficient of wild type protein. Our results support emerging models for a glycan-independent chaperone role for calnexin and for the mechanism of retention of misfolded membrane proteins in the endoplasmic reticulum.     

Functional analysis of the combined role of the O-linked branching enzyme core 2 beta1-6-N-glucosaminyltransferase and dimerization of P-selectin glycoprotein ligand-1 in rolling on P-selectin

Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including alpha1,3-fucosyltransferase-VII, core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more alpha2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys(320)) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength.     

Studying protein dynamics in living cells

Since the advent of the green fluorescent protein, the subcellular localization, mobility, transport routes and binding interactions of proteins can be studied in living cells. Live cell imaging, in combination with photobleaching, energy transfer or fluorescence correlation spectroscopy are providing unprecedented insights into the movement of proteins and their interactions with cellular components. Remarkably, these powerful techniques are accessible to non-specialists using commercially available microscope systems.     

Mechanism of collapse of endoplasmic reticulum cisternae during African swine fever virus infection

Infection of cells with African swine fever virus (ASFV) can lead to the formation of zipper-like stacks of structural proteins attached to collapsed endoplasmic reticulum (ER) cisternae. We show that the collapse of ER cisternae observed during ASFV infection is dependent on the viral envelope protein, J13Lp. Expression of J13Lp alone in cells is sufficient to induce collapsed ER cisternae. Collapse was dependent on a cysteine residue in the N-terminal domain of J13Lp exposed to the ER lumen. Luminal collapse was also dependent on the expression of J13Lp within stacks of ER where antiparallel interactions between the cytoplasmic domains of J13Lp orientated N-terminal domains across ER cisternae. Cisternal collapse was then driven by disulphide bonds between N-terminal domains arranged in antiparallel arrays across the ER lumen. This provides a novel mechanism for biogenesis of modified stacks of ER present in cells infected with ASFV, and may also be relevant to cellular processes.     

Deciphering membrane protein structures from protein sequences

ABSTRACT: Co-evolving positions within protein sequences have been used as spatial constraints to develop a computational approach for modeling membrane protein structures.     

GeneTools – application for functional annotation and statistical hypothesis testing

Background  

Modern biology has shifted from "one gene" approaches to methods for genomic-scale analysis like microarray technology, which allow simultaneous measurement of thousands of genes. This has created a need for tools facilitating interpretation of biological data in "batch" mode. However, such tools often leave the investigator with large volumes of apparently unorganized information. To meet this interpretation challenge, gene-set, or cluster testing has become a popular analytical tool. Many gene-set testing methods and software packages are now available, most of which use a variety of statistical tests to assess the genes in a set for biological information. However, the field is still evolving, and there is a great need for "integrated" solutions.     

Smallholder farms have and can store more carbon than previously estimated

Increasing soil organic carbon (SOC) stocks is increasingly targeted as a key strategy in climate change mitigation and improved ecosystem resiliency. Agricultural land, a dominant global land use, provides substantial challenges and opportunities for global carbon sequestration. Despite this, global estimates of soil carbon sequestration potential often exclude agricultural land and estimates are coarse for regions in the Global South. To address these discrepancies and improve estimates, we develop a hybrid, data-augmented database approach to better estimate the magnitude of SOC sequestration potential of agricultural soils. With high-resolution (30 m) soil maps of Africa developed by the International Soils Database (iSDA) and Malawi as a case study, we create a national adjustment using site-specific soil data retrieved from 1160 agricultural fields. We use a benchmark approach to estimate the amount of SOC Malawian agricultural soils can sequester, accounting for edaphic and climatic conditions, and calculate the resulting carbon gap. Field measurements of SOC stocks and sequestration potentials were consistently larger than iSDA predictions, with an average carbon gap of 4.42 ± 0.23 Mg C ha⁻¹ to a depth of 20 cm, with some areas exceeding 10 Mg C ha⁻¹. Augmenting iSDA predictions with field data also improved sensitivity to identify areas with high SOC sequestration potential by 6%—areas that may benefit from improved management practices. Overall, we estimate that 6.8 million ha of surface soil suitable for agriculture in Malawi has the potential to store 274 ± 14 Tg SOC. Our approach illustrates how ground truthing efforts remain essential to reduce errors in continent-wide soil carbon predictions for local and regional use. This work begins efforts needed across regions to develop soil carbon benchmarks that inform policies and identify high-impact areas in the effort to increase SOC globally.