Improvement of bacterial cellulose production by manipulating the metabolic pathways in which ethanol and sodium citrate involved

Nowadays, bacterial cellulose has played more and more important role as new biological material for food industry and medical and industrial products based on its unique properties. However, it is still a difficult task to improve the production of bacterial cellulose, especially a large number of byproducts are produced in the metabolic biosynthesis processes. To improve bacterial cellulose production, ethanol and sodium citrate are added into the medium during the fermentation, and the activities of key enzymes and concentration of extracellular metabolites are measured to assess the changes of the metabolic flux of the hexose monophosphate pathway (HMP), the Embden–Meyerhof–Parnas pathway (EMP), and the tricarboxylic acid cycle (TCA). Our results indicate that ethanol functions as energy source for ATP generation at the early stage of the fermentation in the HMP pathway and the supplementation of ethanol significantly reduces glycerol generation (a major byproduct). While in the EMP pathway, sodium citrate plays a key role, and its supplementation results in the byproducts (mainly acetic acid and pyruvic acid) entering the gluconeogenesis pathway for cellulose synthesis. Furthermore, by adding ethanol and sodium citrate, the main byproduct citric acid in the TCA cycle is also reduced significantly. It is concluded that bacterial cellulose production can be improved by increasing energy metabolism and reducing the formation of metabolic byproducts through the metabolic regulations of the bypasses.     

真核细胞非经典蛋白分泌途径

在生物体中, 细胞间的信息传递是细胞生长、分化、发育、增殖、凋亡等生命活动的基本保证, 而蛋白分泌是细胞间信息传递的重要方式。大多数分泌蛋白都是通过内质网-高尔基体(ER-Golgi)途径分泌的。然而越来越多的研究表明, 存在着一类无信号肽的分泌蛋白, 这类蛋白不依赖ER-Golgi途径就能分泌到细胞外发挥功能, 被称为非经典分泌蛋白。非经典蛋白的分泌有其特有的机制, 它对ER-Golgi分泌途径是一种必要和有益的补充。非经典分泌与细胞增殖、免疫反应、肿瘤形成、传染病病理学等密切相关。文章旨在对非经典分泌蛋白的特点、分泌机制及生物学意义进行概述。     

Multiple roles of integrins in cell motility

Motility is essential for many important biological events, including embryonic development, inflammatory responses, wound healing, and tumor metastasis. During these events cells are in dynamic contact with the extracellular matrix through integrins. Integrins are the primary receptors for extracellular matrix proteins and consequently are required for cell motility. Cells have evolved multiple mechanisms to modulate integrin adhesive functions, which impact cell migration. In addition to providing a mechanism that allows cells to contact the extracellular matrix, integrins also promote intracellular signals that stimulate and regulate cell movement. Here we discuss the role of integrins during the multiple steps of cell migration.     

MAPK signal specificity: the right place at the right time

Although the mechanisms that lead to activation of the Ras, extracellular-signal-regulated kinase mitogen-activated protein kinase (Ras/ERK-MAPK) signaling pathway have been studied intensively, the fundamental principles that determine how activation of ERK signaling can result in distinct biological outcomes have only recently received attention. Factors such as cell-surface receptor density, expression of scaffolding proteins, the surrounding extracellular matrix, and the interplay between kinases and phosphatases modulate the strength and duration of ERK signaling. Furthermore, the spatial distribution and temporal qualities of ERK can markedly alter the qualitative and quantitative features of downstream signaling to immediate early genes (IEG) and the expression of IEG-encoded protein products. As a result, IEG products provide a molecular interpretation of ERK dynamics, enabling the cell to program an appropriate biological response.     

Mechanical strain modulates extracellular matrix degradation and byproducts in an isoform-specific manner

Many studies have shown that mechanical forces can alter collagen degradation by proteases, and this mechanochemical effect may potentially serve an important role in determining extracellular matrix content and organization in load-bearing tissues. However, it is not yet known whether mechano-sensitive degradation depends on particular protease isoforms, nor is it yet known whether particular degradation byproducts can be altered by mechanical loading. In this study, we tested the hypothesis that different types of proteases exhibit different sensitivities to mechanical loading both in degradation rates and byproducts. Decellularized porcine pericardium samples were treated with human recombinant matrix metalloproteinases-1, ?8, ?9, cathepsin K, or a protease-free control while subjected to different levels of strain in a planar, biaxial mechanical tester. Tissue degradation was monitored by tracking the decay in mechanical stresses during displacement control tests, and byproducts were assessed by mass spectrometry analysis of the sample supernatant after degradation. Our key finding shows that cathepsin K-mediated degradation of collagenous tissue was enhanced with increasing strain, while MMP1-, MMP8-, and MMP9-mediated degradation were first decreased and then increased by strain. Degradation induced changes in tissue mechanical properties, and proteomic analysis revealed strain-sensitive degradome signatures with different ECM byproducts released at low vs. high strains. This evidence suggests a potentially new type of mechanobiology wherein mechanical forces alter the degradation products that can provide important signaling feedback functions during tissue remodeling.     

Functional importance of lysyl oxidase family propeptide regions

The lysyl oxidase family of proteins is primarily known for its critical role in catalyzing extracellular oxidative deamination of hydroxylysine and lysine residues in collagens, and lysine residues in elastin required for connective tissue structure and function. Lysyl oxidases have additional important biological functions in health and disease. While the enzyme domains are highly conserved, the propeptide regions are less uniform, and have biological activity, some of which are independent of their respective enzymes. This review summarizes what has been published regarding the functions of the propeptide regions of this family of proteins in the context of extracellular matrix biosynthesis, fibrosis and cancer biology. Although much has been learned, there is a need for greater attention to structure/function relationships and mechanisms to more fully understand these multifunctional proteins.     

Type IV collagenases (MMP-2 and MMP-9) and their substrates--intracellular proteins, hormones, cytokines, chemokines and their receptors

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave protein components of extracellular matrix such as collagens, laminin, fibronectin, proteoglycans and contribute to cell migration by eliminating the surrounding extracellular matrix and basement membrane barriers. However, the extracellular matrix is not simply an extracellular scaffold because, for example, it contains sites that can bind growth factors; therefore, degradation of the extracellular matrix components by MMPs can alter cellular behavior. MMPs also cleave a variety of non-ECM proteins, including cytokines, chemokines, and growth factors, activating or inactivating them, or generating other products that have biological consequences. The immune system is also influenced by MMPs. For that reason, the function of MMPs is much more complex and subtle than simple demolition. MMPs are essential for embryonic development and morphogenesis, however, exuberant expression of these enzymes has been associated with a variety of destructive diseases, including tumor progression, cardiovascular diseases and autoimmune diseases.     

Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes.

How intracellular cytoskeletal and signaling proteins connect and communicate with the extracellular matrix (ECM) is a fundamental question in cell biology. Recent biochemical, cell biological, and genetic studies have revealed important roles of cytoplasmic integrin-linked kinase (ILK) and its interactive proteins in these processes. Cell adhesion to ECM is an important process that controls cell shape change, migration, proliferation, survival, and differentiation. Upon adhesion to ECM, integrins and a selective group of cytoskeletal and signaling proteins are recruited to cell matrix contact sites where they link the actin cytoskeleton to the ECM and mediate signal transduction between the intracellular and extracellular compartments. In this review, we discuss the molecular activities and cellular functions of ILK, a protein that is emerging as a key component of the cell-ECM adhesion structures.     

Modulating the Mechanical Stability of Extracellular Matrix Protein Tenascin-C in a Controlled and Reversible Fashion

Stretching force can induce conformational changes of proteins and is believed to be an important biological signal in the mechanotransduction network. Tenascin-C is a large extracellular matrix protein and is subject to stretching force under its physiological condition. Regulating the mechanical properties of the fibronectin type III domains of tenascin-C will alter its response to mechanical stretching force and thus may provide the possibility of regulating the biological activities of tenascin-C in living cells. However, tuning the mechanical stability of proteins in a rational and systematic fashion remains challenging. Using the third fibronectin type III domain (TNfn3) of tenascin-C as a model system, here we report a successful engineering of a mechanically stronger extracellular matrix protein via engineered metal chelation. Combining steered molecular dynamics simulations, protein engineering and single-molecule atomic force microscopy, we have rationally engineered a bihistidine-based metal chelation site into TNfn3. We used its metal chelation capability to selectively increase the unfolding energy barrier for the rate-limiting step during the mechanical unfolding of TNfn3. The resultant TNfn3 mutant exhibits enhanced mechanical stability. Using a stronger metal chelator, one can convert TNfn3 back to a state of lower mechanical stability. This is the first step toward engineering extracellular matrix proteins with defined mechanical properties, which can be modulated reversibly by external stimuli, and will provide the possibility of using external stimuli to regulate the biological functions of extracellular matrix proteins.     

Hyaluronan catabolism: a new metabolic pathway

A new pathway of intermediary metabolism is described involving the catabolism of hyaluronan. The cell surface hyaluronan receptor, CD44, two hyaluronidases, Hyal-1 and Hyal-2, and two lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase, are involved. This metabolic cascade begins in lipid raft invaginations at the cell membrane surface. Degradation of the high-molecular-weight extracellular hyaluronan occurs in a series of discreet steps generating hyaluronan chains of decreasing sizes. The biological functions of the oligomers at each quantum step differ widely, from the space-filling, hydrating, anti-angiogenic, immunosuppressive 10(4)-kDa extracellular polymer, to 20-kDa intermediate polymers that are highly angiogenic, immuno-stimulatory, and inflammatory. This is followed by degradation to small oligomers that can induce heat shock proteins and that are anti-apoptotic. The single sugar products, glucuronic acid and a glucosamine derivative are released from lysosomes to the cytoplasm, where they become available for other metabolic cycles. There are 15 g of hyaluronan in the 70-kg individual, of which 5 g are cycled daily through this pathway. Some of the steps in this catabolic cascade can be commandeered by cancer cells in the process of growth, invasion, and metastatic spread.     

Cumulus oophorus extracellular matrix: its construction and regulation.

Cumulus oophorus, an investing structure unique to oocytes of higher mammals, is induced to synthesize an extensive extracellular matrix by ovulatory stimulus, leading to the characteristic preovulatory expansion of the cumulus-oocyte complex. The extracellular matrix consists of cumulus cell-secreted hyaluronan, proteoglycans and proteins, as well as extrafollicularly originated SHAPs (serum-derived hyaluronan-associated proteins) that are bound covalently to hyaluronan. The secretion and assembly of matrix molecules by cumulus cells are temporally regulated by factors derived from both mural granulosa cells and oocyte, which synchronize the deposition of the cumulus oophorus matrix with other intrafollicular ovulatory events. The cumulus oophorus matrix is essential for ovulation and subsequent fertilization. Recently, taking advantage of animal models with defined genetic modifications, it has become possible to investigate in vivo the structure of the cumulus oophorus matrix, the regulatory mechanism for matrix deposition and its biological functions. This review focuses on the recent findings on the construction of the cumulus oophorus matrix and the regulation.     

Proteome analysis of matrix vesicles isolated from femurs of chicken embryo

Matrix vesicles (MVs) are extracellular organelles that initiate mineral formation, accumulating inorganic phosphate (P(i)) and calcium leading to the formation of hydroxyapatite (HA) crystals, the main mineral component of bones. MVs are produced during bone formation, as well as during the endochondral calcification of cartilage. MVs are released into the extracellular matrix from osseous cells such as osteoblasts and hypertrophic chondrocytes. In this report, using 1-D SDS-PAGE, in-gel tryptic digestion and an LC-MS-MS/MS protein identification protocol, we characterized the proteome of MVs isolated from chicken embryo (Gallus gallus) bones and cartilage. We identified 126 gene products, including proteins related to the extracellular matrix and ion transport, as well as enzymes, cytoskeletal, and regulatory proteins. Among the proteins recognized for the first time in MVs were aquaporin 1, annexin A1 (AnxA1), AnxA11, glycoprotein HT7, G(i) protein alpha2, and scavenger receptor type B. The pathways for targeting the identified proteins into MVs and their particular functions in the biomineralization process are discussed. Obtaining a knowledge of the functions and roles of these proteins during embryonic mineralization is a prerequisite for the overall understanding of the initial mineral formation mechanisms.     

Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis

Epithelial cells must adhere to the extracellular matrix (ECM) for survival, as detachment from matrix triggers apoptosis or anoikis. Integrins are major mediators of adhesion between cells and ECM proteins, and transduce signals required for cell survival. Recent evidence suggests that integrin receptors are coupled to growth factor receptors in the regulation of multiple biological functions; however, mechanisms involved in coordinate regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we identify the pro-apoptotic protein Bim as a critical mediator of anoikis in epithelial cells. Bim is strongly induced after cell detachment and downregulation of Bim expression by RNA interference (RNAi) inhibits anoikis. Detachment-induced expression of Bim requires a lack of beta(1)-integrin engagement, downregulation of EGF receptor (EGFR) expression and inhibition of Erk signalling. Overexpressed EGFR was uncoupled from integrin regulation, resulting in the maintenance of Erk activation in suspension, and a block in Bim expression and anoikis. Thus, Bim functions as a key sensor of integrin and growth factor signals to the Erk pathway, and loss of such coordinate regulation may contribute to tumour progression.     

Hyaluronan: a multifunctional, megaDalton, stealth molecule

Hyaluronan has been implicated in biological processes such as cell adhesion, migration and proliferation. Traditionally, it was thought to be associated with the extracellular matrix, but, hyaluronan may also have unimagined roles inside the cell. Investigation of hyaluronan synthesis and degradation, the identification of new receptors and binding proteins, and the elucidation of hyaluronan-dependent signaling pathways are providing novel insights into the true biological functions of this fascinating molecule.     

The collagen family

Collagens are the most abundant proteins in mammals. The collagen family comprises 28 members that contain at least one triple-helical domain. Collagens are deposited in the extracellular matrix where most of them form supramolecular assemblies. Four collagens are type II membrane proteins that also exist in a soluble form released from the cell surface by shedding. Collagens play structural roles and contribute to mechanical properties, organization, and shape of tissues. They interact with cells via several receptor families and regulate their proliferation, migration, and differentiation. Some collagens have a restricted tissue distribution and hence specific biological functions.     

Glycosaminoglycan interaction networks and databases

Glycosaminoglycans are complex polysaccharides exhibiting a large structural and conformational diversity. These key biological players organize the extracellular matrix, contribute to cell–matrix interactions, and regulate cell signaling. Natural and synthetic libraries of glycosaminoglycans have been spotted on microarrays to find glycosaminoglycan partners and determine the size and the chemical groups promoting protein binding. Advances in glycosaminoglycan sequencing allow the characterization of glycosaminoglycan sequences interacting with proteins, and glycosaminoglycan-mediated pull-down proteomics can identify glycosaminoglycan-binding proteins at a proteome scale in various biological samples. The analysis of the glycosaminoglycan interaction networks generated using these data gives insights into the molecular and cellular mechanisms underlying glycosaminoglycan functions. These interactomes can also be used to design inhibitors targeting specific GAG interactions for therapeutic purpose.     

Making recombinant extracellular matrix proteins

A variety of approaches to understand extracellular matrix protein structure and function require production of recombinant proteins. Moreover, the expression of heterologous extracellular matrix proteins, in particular collagens, using the recombinant technology is of major interest to the biomedical industry. Although extracellular matrix proteins are large, modular and often multimeric, most of them have been successfully produced in various expression systems. This review provides important factors, including the design of the construct, the cloning strategies, the expression vectors, the transfection method and the host cell systems, to consider in choosing a reliable and cost-effective way to make recombinant extracellular matrix proteins. Advantages and drawbacks of each system have been appraised. Protocols that may ease efficient recombinant production of extracellular matrix are described. Emphasis is placed on the recombinant collagen production. Members of the collagen superfamily exhibit specific structural features and generally require complex post-translational modifications to retain full biological activity that make more arduous their recombinant production.     

Altered amelogenin self-assembly based on mutations observed in human X-linked amelogenesis imperfecta (AIH1)

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin, a 180-amino acid hydrophobic protein that self-assembles to form nanospheres. We have used independent technical methods, consisting of the yeast two-hybrid (Y2H) assay and surface plasmon resonance (SPR), to demonstrate the importance of amelogenin self-assembly domains. In addition, we have analyzed mutations in amelogenin observed in patients with amelogenesis imperfecta who demonstrate defects in enamel formation. Assessments of self-assembly of these mutant amelogenins by either SPR or Y2H assay yield concordant data. These data support the conclusion that the amelogenin amino-terminal self-assembly domain is essential to the creation of an enamel extracellular organic matrix capable of directing mineral formation. It also suggests that a pathway through which point mutations in the amelogenin protein can adversely impact on the formation of the enamel organ is by disturbing self-assembly of the organic matrix. These data support the utilization of the Y2H assay to search for protein interactions among extracellular matrix proteins that contribute to biomineralization and provide functional information on protein-protein and protein-mineral interactions.     

PI3K/Akt信号通路相关的生物学调控机制研究进展

PI3K/Akt信号通路是由酶联受体介导的信号转导通路,该通路不仅参与多种生长因子、细胞因子和细胞外基质等的信号转导,同时还参与细胞增殖、分化、凋亡和葡萄糖转运等多种细胞功能的调节,特别是在细胞凋亡、细胞存活以及调控细胞糖代谢等方面具有重要作用。本研究综述了PI3K-Akt信号通路的结构组成、通路活化、通信过程、调控机制及其生物学功能等方面的研究进展,为进一步研究PI3K/Akt信号通路的生物学调控作用机制提供启示。