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Yeast expression systems have been successfully used for over 20 years for the production of recombinant proteins. With the growing interest in recombinant protein expression for various uses, yeast expression systems, such as the popular Pichia pastoris, are becoming increasingly important. Although P pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, there is still room for improvement of this expression system. In particular, secretion of recombinant proteins is still one of the main reasons for using P pastoris. Therefore, endoplasmic reticulum protein folding, correct glycosylation, vesicular transport to the plasma membrane, gene dosage, secretion signal sequences, and secretome studies are important considerations for improved recombinant protein production. [PUBLICATION ABSTRACT]

Distinct pathways for protein secretion have been revealed in plant cells, both conventional and unconventional. Advanced techniques have been developed to unveil their underlying mechanisms. Abstract Protein secretion is an essential process in all eukaryotic cells and its mechanisms have been extensively studied. Proteins with an N-terminal leading sequence or transmembrane domain are delivered through the conventional protein secretion (CPS) pathway from the endoplasmic reticulum (ER) to the Golgi apparatus. This feature is conserved in yeast, animals, and plants. In contrast, the transport of leaderless secretory proteins (LSPs) from the cytosol to the cell exterior is accomplished via the unconventional protein secretion (UPS) pathway. So far, the CPS pathway has been well characterized in plants, with several recent studies providing new information about the regulatory mechanisms involved. On the other hand, studies on UPS pathways in plants remain descriptive, although a connection between UPS and the plant defense response is becoming more and more apparent. In this review, we present an update on CPS and UPS. With the emergence of new techniques, a more comprehensive understanding of protein secretion in plants can be expected in the future.

Staphylococcus aureus is a leading cause of infections worldwide. S. aureus utilizes a specialized type VIIb secretion system (T7SSb) to persist in the infected host tissues as well as target competitor bacteria to establish its niche. T7SSb assembles into a multiprotein translocation complex and facilitates secretion of a set of small proteins and larger polymorphic toxins across the cytosolic membrane. Beyond the membrane, secreted proteins were thought to diffuse through the thick yet porous cell wall and release into the environment. Here, we demonstrate for the first time that S. aureus T7SSb extends across the cell wall via its EsaA subunit. Furthermore, accommodation of EsaA within the cell wall requires an associated cell wall hydrolase EssH and is essential for protein secretion via T7SSb. Thus, our findings provide a mechanistic insight for a coordinated cell wall processing and T7SSb assembly to support specialized protein secretion in S. aureus .

How cells determine where to assemble a macromolecular complex is a fundamental question in biology since the localization of these complexes is directly linked to functions. In bacteria, the type VI secretion system (T6SS) relies on effective positioning to target competitor and host cells in contact-dependent interactions. This study identifies a PpkA-TssW-Fha axis that orchestrates T6SS localization and activation through membrane anchoring and liquid-liquid phase separation at the inner membrane interface. These new insights can help us not only better understand how the T6SS functions but also better design T6SS-based solutions for therapeutic targeting of drug-resistant and T6SS-susceptible bacterial and fungal pathogens.

Bordetella bronchiseptica and Bordetella pertussis are two closely related respiratory pathogens that employ their T3SS injectisome to deliver the BteA effector into host cells. In this study, we visualized the needle tip filament of their T3SS injectisome, a structure formed by the Bsp22 protein. We demonstrate that during Bordetella cultivation in Stainer-Scholte medium, Bsp22 filaments are abundant and can dynamically extend up to several micrometers in length through the incorporation of new subunits at their distal ends. In contrast, these filaments become shorter and/or less abundant during infection of host cells. This reduction correlates with decreased bsp22 mRNA expression and lower Bsp22 protein levels, while the levels of bscD mRNA, which encodes the inner membrane ring protein of the injectisome, remain stable. These results highlight the adaptability of the Bordetella T3SS injectisome and show how its tip filament structure changes in response to different environments.

Protein secretion is a common property of pathogenic microbes. Gram-negative bacterial pathogens use at least 6 distinct extracellular protein secretion systems to export proteins through their multilayered cell envelope and in some cases into host cells. Among the most widespread is the newly recognized Type VI secretion system (T6SS) which is composed of 15-20 proteins whose biochemical functions are not well understood. Using crystallographic, biochemical, and bioinformatic analyses, we identified 3 T6SS components, which are homologous to bacteriophage tail proteins. These include the tail tube protein; the membrane-penetrating needle, situated at the distal end of the tube; and another protein associated with the needle and tube. We propose that T6SS is a multicomponent structure whose extracellular part resembles both structurally and functionally a bacteriophage tail, an efficient machine that translocates proteins and DNA across lipid membranes into cells.

Cancer-associated fibroblasts (CAFs) drive tumour progression, but the emergence of this cell state is poorly understood. A broad spectrum of metalloproteinases, controlled by the Timp gene family, influence the tumour microenvironment in human cancers. Here, we generate quadruple TIMP knockout (TIMPless) fibroblasts to unleash metalloproteinase activity within the tumour-stromal compartment and show that complete Timp loss is sufficient for the acquisition of hallmark CAF functions. Exosomes produced by TIMPless fibroblasts induce cancer cell motility and cancer stem cell markers. The proteome of these exosomes is enriched in extracellular matrix proteins and the metalloproteinase ADAM10. Exosomal ADAM10 increases aldehyde dehydrogenase expression in breast cancer cells through Notch receptor activation and enhances motility through the GTPase RhoA. Moreover, ADAM10 knockdown in TIMPless fibroblasts abrogates their CAF function. Importantly, human CAFs secrete ADAM10-rich exosomes that promote cell motility and activate RhoA and Notch signalling in cancer cells. Thus, Timps suppress cancer stroma where activated-fibroblast-secreted exosomes impact tumour progression. Khokha and colleagues report that loss of the Timp family of metalloproteinases from stromal fibroblasts promotes a cancer-associated fibroblast phenotype and production of exosomes that stimulate cancer cell motility.

Salmonella enterica is an increasing global public health threat. As part of its virulence arsenal, Salmonella relies on a type III secretion system (T3SS) or injectisome, a molecular injection device that translocates effector proteins into host cells to promote invasion and inflammation. A central component of this machine is the SpaO protein, which is produced in two forms: a full-length form and a shorter variant. Here, by studying the functional and structural relationship between the two SpaO forms in their native cellular environment, we define how and when they assemble within the injectisome. Employing quantitative injection assays in cultured cells, we define the shorter SpaO variant as an accessory structural piece that boosts effector delivery. These findings refine our understanding of injectisome assembly and function and provide mechanistic insight to inform future efforts to target T3SS-dependent pathogens through antivirulence strategies.

Summary Microalga‐based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post‐translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C‐terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n, wherein n = 10 or 20]. The yields of the (SP)n‐fused Venus were higher than Venus without the glycomodule by up to 12‐fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus‐(SP)n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae‐based recombinant proteins.

Plants are promising next‐generation hosts for recombinant protein production; however, major challenges remain with regard to enhancing the efficiency of downstream processing, particularly in the removal of cellular residues and purification of the expressed proteins. Strategies to overcome these limitations include targeting expressed recombinant proteins within a specific organelle or directing their secretion into the extracellular space, thereby facilitating purification by collecting the target matrix. In this study, we focused on protein secretion mechanisms and identified two pathogenesis‐related proteins, glucan endo‐1,3‐β‐glucosidase (GN) and chitinase 8 (Chi8), which accumulated in the apoplast washing fluid following Agrobacterium infiltration of Nicotiana benthamiana leaves. Both proteins contained signal peptides (SPs), SPGN and SPChi8, respectively. Although the intracellular accumulation of GFP was comparable regardless of the expression level, fusion with either SPGN or SPChi8 resulted in GFP accumulation within the apoplast. In contrast, in N. benthamiana, a mammalian‐derived SP was less effective in facilitating GFP secretion than the plant‐derived SPs. Additionally, replacing the SP of the mammalian‐derived protein β‐glucocerebrosidase (GCase) with SPGN or SPChi8 enhanced the secretion of GCase into the apoplast, indicating their applicability in protein production. Moreover, SPGN and SPChi8 directed the expressed proteins into the culture medium of N. benthamiana suspension cells. These results indicate that SPGN and SPChi8 function as effective secretion signals and highlight the potential application of endogenous SPs for enhancing recombinant protein production in plants.