Publicaciones

ABSTRACT

Phospholipid flippases (P4-ATPases) are central to the establishment of membrane lipid asymmetry, a property underlying membrane trafficking and polarized growth in eukaryotic cells. However, the specific contribution of individual flippases to hyphal morphogenesis in filamentous fungi remains poorly defined. Here, we characterize DNF-4, a putative P4-ATPase in Neurospora crassa with high sequence identity to the essential flippase Neo1 of Saccharomyces cerevisiae. Using endogenous tagging and live-cell imaging, we show that DNF-4 localizes to highly dynamic punctate structures associated with endoplasmic reticulum- and Golgi-related compartments, supported by quantitative co-localization analyses with the ER marker CSE-7-mChFP and the Golgi-associated Rab GTPase YPT-1-mChFP. FRAP experiments revealed partial fluorescence recovery, indicating dynamic exchange of DNF-4- associated compartments. These structures undergo bidirectional movement along the hypha, and their motility is strongly dependent on an intact microtubule cytoskeleton. Deletion of dnf-4 results in pronounced defects in hyphal growth and development, including reduced hyphal elongation, decreased biomass accumulation, smaller conidia, and a severe reduction in conidiation. These defects are accompanied by increased branching frequency, abnormal hyphal morphology, and altered Spitzenkorper ¨ positioning and dynamics, indicating impaired coordination of polarized growth. Our results demonstrate that DNF-4 contributes to membrane trafficking processes required for the maintenance of hyphal polarity and normal developmental progression in N. crassa. These findings provide new evidence that P4-ATPases play an important role in the spatial organization of membrane trafficking pathways underlying fungal morphogenesis.

KEYWORDS
Neurospora crassa, Phospholipid flippase, DNF-4, Polarized growth, Membrane trafficking, Conidiation, Spitzenkorper 

BACKGROUND

Chagas disease, caused by the intracellular parasite Trypanosoma cruzi, is one of the most important parasitological infections in the Americas. It is estimated to infect approximately 6 million people from mostly low income countries in Latin America, although recent infections have been reported in southern US states. Several studies have described an extensive genetic diversity among T. cruzi isolates throughout its geographic distribution in the American continent. This diversity has been correlated with the pathology developed during an infection. However, due to a lack of a single reliable test, current diagnosis practices of the disease are not straightforward since several different tests are applied. The use of current genomic sequence data allows for the selection of molecular markers (MM) that have the ability to identify the Discrete Typing Unit (DTU) of T. cruzi in a given infection, without the need of any sequencing reaction

BACKGROUND

Protein solubility characteristics are important determinants of success for recombinant proteins in relation to expression, purification, storage and administration. Escherichia coli offers a cost-efficient expression system. An important limitation, whether for biophysical studies or industrial-scale production, is the formation of insoluble protein aggregates in the cytoplasm. Several strategies have been implemented to improve soluble expression, ranging from modification of culture conditions to inclusion of solubility-enhancing tags.

RESULTS

Surface patch analysis has been applied to predict amino acid changes that can alter the solubility of expressed recombinant human erythropoietin (rHuEPO) in E. coli, a factor that has importance for both yield and subsequent downstream processing of recombinant proteins. A set of rHuEPO proteins (rHuEPO E13K, F48D, R150D, and F48D/R150D) was designed (from the framework of wild-type protein, rHuEPO WT, via amino acid mutations) that varied in terms of positively-charged patches. A variant predicted to promote aggregation (rHuEPO E13K) decreased solubility significantly compared to rHuEPO WT. In contrast, variants predicted to diminish aggregation (rHuEPO F48D, R150D, and F48D/R150D) increased solubility up to 60% in relation to rHuEPO WT.

KEYWORDS

Abstract

Due in part to the needs of the biopharmaceutical industry, there has been an increased drive to generate high quality recombinant proteins in large amounts. However, achieving high yields can be a challenge as the novelty and increased complexity of new targets often makes them ‘difficult‐to‐express’. This study aimed to define the molecular features that restrict the production of a model ‘difficult‐to‐express’ recombinant protein, Tissue Inhibitor Metalloproteinase‐3 (TIMP‐3). Building from experimental data, computational approaches were used to rationalize the redesign of this recombinant target to generate a chimera with enhanced secretion. The results highlight the importance of early identification of unfavourable sequence attributes, enabling the generation of engineered protein forms that bypass ‘secretory’ bottlenecks and result in efficient recombinant protein production.

KEYWORDS

 Difficult‐to‐express, mammalian expression system, predictive computational tool, protein engineering, recombinant protein production, tissue inhibitor of metalloproteinase

MOTIVATION

Protein solubility is an important property in industrial and therapeutic applications. Prediction is a challenge, despite a growing understanding of the relevant physicochemical properties.

RESULTS

Protein-Sol is a web server for predicting protein solubility. Using available data for Escherichia coli protein solubility in a cell-free expression system, 35 sequence-based properties are calculated. Feature weights are determined from separation of low and high solubility subsets. The model returns a predicted solubility and an indication of the features which deviate most from average values. Two other properties are profiled in windowed calculation along the sequence: fold propensity, and net segment charge. The utility of these additional features is demonstrated with the example of thioredoxin.