Four thiol-oxidoreductases involved in the formation of disulphide bonds in the Streptomyces lividans TK21 secretory proteins

Native disulphide bonds are essential for the structure and function of many membrane and secretory proteins. Disulphide bonds are formed, reduced and isomerized in the endoplasmic reticulum of mammalian cells by a family of oxidoreductases, which includes protein disulphide isomerase (PDI), ERp57, ERp72, P5 and PDIR. This review will discuss how these enzymes are maintained in either an oxidized redox state that allows them to form disulphide bonds in substrate proteins or a reduced form that allows them to perform isomerization and reduction reactions, how these opposing pathways may co-exist within the same compartment and why so many oxidoreductases exist when PDI alone can perform all three of these functions.

Download Full-text

Dynamic metabolic modelling of overproduced protein secretion in Streptomyces lividans using adaptive DFBA

BMC Microbiology ◽

10.1186/s12866-019-1591-7 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Jósé R. Valverde ◽

Sonia Gullón ◽

Clara A. García-Herrero ◽

Iván Campoy ◽

Rafael P. Mellado

Keyword(s):

Protein Secretion ◽

Environmental Changes ◽

Streptomyces Lividans ◽

Secretory Protein ◽

Time Dependent ◽

Secretory Proteins ◽

Exogenous Dna ◽

Dynamic Flux Balance Analysis ◽

Balance Analysis ◽

Living Organisms

Abstract Background Streptomyces lividans is an appealing host for the production of proteins of biotechnological interest due to its relaxed exogenous DNA restriction system and its ability to secrete proteins directly to the medium through the major Sec or the minor Tat routes. Often, protein secretion displays non-uniform time-dependent patterns. Understanding the associated metabolic changes is a crucial step to engineer protein production. Dynamic Flux Balance Analysis (DFBA) allows the study of the interactions between a modelled organism and its environment over time. Existing methods allow the specification of initial model and environment conditions, but do not allow introducing arbitrary modifications in the course of the simulation. Living organisms, however, display unexpected adaptive metabolic behaviours in response to unpredictable changes in their environment. Engineering the secretion of products of biotechnological interest has systematically proven especially difficult to model using DFBA. Accurate time-dependent modelling of complex and/or arbitrary, adaptive metabolic processes demands an extended approach to DFBA. Results In this work, we introduce Adaptive DFBA, a novel, versatile simulation approach that permits inclusion of changes in the organism or the environment at any time in the simulation, either arbitrary or interactively responsive to environmental changes. This approach extends traditional DFBA to allow steering arbitrarily complex simulations of metabolic dynamics. When applied to Sec- or Tat-dependent secretion of overproduced proteins in S. lividans, Adaptive DFBA can overcome the limitations of traditional DFBA to reproduce experimental data on plasmid-free, plasmid bearing and secretory protein overproducing S. lividans TK24, and can yield useful insights on the behaviour of systems with limited experimental knowledge such as agarase or amylase overproduction in S. lividans TK21. Conclusions Adaptive DFBA has allowed us to overcome DFBA limitations and to generate more accurate models of the metabolism during the overproduction of secretory proteins in S. lividans, improving our understanding of the underlying processes. Adaptive DFBA is versatile enough to permit dynamical metabolic simulations of arbitrarily complex biotechnological processes.

Download Full-text

Streptomyces lividans contains a minimal functional signal recognition particle that is involved in protein secretion

Microbiology ◽

10.1099/mic.0.26313-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2435-2442 ◽

Cited By ~ 20

Author(s):

Arantxa Palacín ◽

Ricardo de la Fuente ◽

Inmaculada Valle ◽

Luis A. Rivas ◽

Rafael P. Mellado

Keyword(s):

Protein Secretion ◽

Signal Recognition Particle ◽

Streptomyces Lividans ◽

Secretory Proteins ◽

Signal Recognition

The bacterial version of the mammalian signal recognition particle (SRP) is well conserved and essential to all known bacteria. The genes for the Streptomyces lividans SRP components have been cloned and characterized. FtsY resembles the mammalian SRP receptor and the S. lividans SRP consists of Ffh, a homologue of the mammalian SRP54 protein, and scRNA, which is a small size RNA of 82 nt in length. Co-immunoprecipitation studies confirmed that Ffh and scRNA are probably the only components of the S. lividans SRP and that pre-agarase can co-immunoprecipitate with Ffh, suggesting that the SRP is involved in targeting secretory proteins.

Download Full-text

Functional identification of a Streptomyces lividans FKBP-like protein involved in the folding of overproduced secreted proteins

Open Biology ◽

10.1098/rsob.190201 ◽

2019 ◽

Vol 9 (10) ◽

pp. 190201

Author(s):

R. L. Vicente ◽

S. Marín ◽

J. R. Valverde ◽

C. Palomino ◽

R. P. Mellado ◽

...

Keyword(s):

Protein Folding ◽

Secretory Pathway ◽

Streptomyces Lividans ◽

Secretory Protein ◽

Industrial Applications ◽

Secreted Proteins ◽

Secretory Proteins ◽

Heterologous Proteins ◽

Functional Identification ◽

Ppiase Activity

Some bacterial peptidyl-prolyl cis/trans isomerases (PPIases) are involved in secretory protein folding after the translocation step. Streptomyces lividans has been used as a host for engineering extracellular overproduction of homologous and heterologous proteins in industrial applications. Although the mechanisms governing the major secretory pathway (Sec route) and the minor secretory pathway (Tat route) are reasonably well described, the function of proteins responsible for the extracellular secretory protein folding is not characterized as yet. We have characterized a Tat-dependent S . lividans FK506-binding protein-like lipoprotein (FKBP) that has PPIase activity. A mutant in the sli-fkbp gene induces a secretion stress response and affects secretion and activity of the Sec-dependent protein α-amylase. Additionally, propagation in high copy number of the sli-fkbp gene has a positive effect on the activity of both the overproduced α-amylase and the overproduced Tat-dependent agarase, both containing proline cis isomers. Targeted proteomic analyses showed that a relevant group of secreted proteins in S. lividans TK21 are affected by Sli-FKBP, revealing a wide substrate range. The results obtained indicate that, regardless of the secretory route used by proteins in S. lividans , adjusting the expression of sli-fkbp may facilitate folding of dependent proteins when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins.

Download Full-text

Russell bodies: a general response of secretory cells to synthesis of a mutant immunoglobulin which can neither exit from, nor be degraded in, the endoplasmic reticulum.

The Journal of Cell Biology ◽

10.1083/jcb.115.4.983 ◽

1991 ◽

Vol 115 (4) ◽

pp. 983-994 ◽

Cited By ~ 92

Author(s):

C Valetti ◽

C E Grossi ◽

C Milstein ◽

R Sitia

Keyword(s):

Protein S ◽

Myeloma Cell ◽

Glioma Cell Line ◽

Secretory Cells ◽

Chain Gene ◽

Secretory Proteins ◽

Rat Glioma ◽

Disulphide Bonds ◽

Golgi Compartment ◽

General Response

Dilated cisternae of the ER resembling Russell Bodies (RBs) are induced in light (L) chain producing myeloma cell lines by transfection of a mu heavy (H) chain gene lacking the first constant domain (mu delta CH1). RBs do not appear to be tissue specific, since they are also induced in a rat glioma cell line transfected with mu delta CH1 and L chain genes. Efficient RB biogenesis requires H-L assembly and polymerization. The mutant Ig is partially degraded in a pre-Golgi compartment. The remnant, however, becomes an insoluble lattice when intersubunit disulphide bonds are formed. The resulting insoluble aggregate accumulates in RBs. Replacing the COOH-terminal cysteine of mu delta CH1 chains with alanine reverses the RB-phenotype: the double mutant mu ala delta CH1 chains assemble noncovalently with L and are secreted as H2L2 complexes. Similarly, secretion of mu delta CH1 chains can be induced by culturing transfectant cells in the presence of reducing agents. The presence of RBs does not alter transport of other secretory or membrane molecules, nor does it affect cell division. Resident proteins of the ER and other secretory proteins are not concentrated in RBs, implying sorting at the ER level. Sorting could be the result of the specific molecular structure of the insoluble lattice. We propose that RBs represent a general response of the cell to the accumulation of abundant, nondegradable protein(s) that fail to exit from the ER.

Download Full-text

Different redox sensitivity of endoplasmic reticulum associated degradation clients suggests a novel role for disulphide bonds in secretory proteins

Biochemistry and Cell Biology ◽

10.1139/bcb-2013-0090 ◽

2014 ◽

Vol 92 (2) ◽

pp. 113-118 ◽

Cited By ~ 10

Author(s):

Iria Medraño-Fernandez ◽

Claudio Fagioli ◽

Alexandre Mezghrani ◽

Mieko Otsu ◽

Roberto Sitia

Keyword(s):

Endoplasmic Reticulum ◽

Reduced Form ◽

Secretory Proteins ◽

Endoplasmic Reticulum Associated Degradation ◽

Disulphide Bonds ◽

Complex Process ◽

Cargo Proteins ◽

Redox Sensitivity ◽

Partially Unfolded ◽

The Stability

To maintain proteostasis in the endoplasmic reticulum (ER), terminally misfolded secretory proteins must be recognized, partially unfolded, and dislocated to the cytosol for proteasomal destruction, in a complex process called ER-associated degradation (ERAD). Dislocation implies reduction of inter-chain disulphide bonds. When in its reduced form, protein disulphide isomerase (PDI) can act not only as a reductase but also as an unfoldase, preparing substrates for dislocation. PDI oxidation by Ero1 favours substrate release and transport across the ER membrane. Here we addressed the redox dependency of ERAD and found that DTT stimulates the dislocation of proteins with DTT-resistant disulphide bonds (i.e., orphan Ig-μ chains) but stabilizes a ribophorin mutant (Ri332) devoid of them. DTT promotes the association of Ri332, but not of Ig-µ, with PDI. This discrepancy may suggest that disulphide bonds in cargo proteins can be utilized to oxidize PDI, hence facilitating substrate detachment and degradation also in the absence of Ero1. Accordingly, Ero1 silencing retards Ri332 degradation, but has little if any effect on Ig-µ. Thus, some disulphides can increase the stability and simultaneously favour quality control of secretory proteins.

Download Full-text

Faculty Opinions recommendation of Different redox sensitivity of endoplasmic reticulum associated degradation clients suggests a novel role for disulphide bonds in secretory proteins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718339498.793493645 ◽

2014 ◽

Author(s):

Karin Romisch

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Proteins ◽

Endoplasmic Reticulum Associated Degradation ◽

Disulphide Bonds ◽

Redox Sensitivity

Download Full-text

Surface plasmon resonance-based interaction studies reveal competition of Streptomyces lividans type I signal peptidases for binding preproteins

Microbiology ◽

10.1099/mic.0.28734-0 ◽

2006 ◽

Vol 152 (5) ◽

pp. 1441-1450 ◽

Cited By ~ 9

Author(s):

Nick Geukens ◽

Smitha Rao C. V. ◽

Rafael P. Mellado ◽

Filip Frederix ◽

Gunter Reekmans ◽

...

Keyword(s):

Substrate Specificity ◽

Streptomyces Lividans ◽

Substrate Preference ◽

Secretory Proteins ◽

Type I ◽

Processing Level ◽

Signal Peptidases ◽

High Level

Type I signal peptidases (SPases) are responsible for the cleavage of signal peptides from secretory proteins. Streptomyces lividans contains four different SPases, denoted SipW, SipX, SipY and SipZ, having at least some differences in their substrate specificity. In this report in vitro preprotein binding/processing and protein secretion in single SPase mutants was determined to gain more insight into the substrate specificity of the different SPases and the underlying molecular basis. Results indicated that preproteins do not preferentially bind to a particular SPase, suggesting SPase competition for binding preproteins. This observation, together with the fact that each SPase could process each preprotein tested with a similar efficiency in an in vitro assay, suggested that there is no real specificity in substrate binding and processing, and that they are all actively involved in preprotein processing in vivo. Although this seems to be the case for some proteins tested, high-level secretion of others was clearly dependent on only one particular SPase demonstrating clear differences in substrate preference at the in vivo processing level. Hence, these results strongly suggest that there are additional factors other than the cleavage requirements of the enzymes that strongly affect the substrate preference of SPases in vivo.

Download Full-text

Ultrastructural Aspects of Golgi Complex Function in Parathyroid Cells of Winter Frogs

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073842 ◽

1973 ◽

Vol 31 ◽

pp. 680-681

Author(s):

William J. Dougherty

Keyword(s):

Golgi Complex ◽

Secretory Granules ◽

Complex Function ◽

Secretory Activity ◽

Secretory Proteins ◽

Perivascular Spaces ◽

Pituitary Cells ◽

Electron Microscopic ◽

Ca Ions ◽

Regulation Of Secretion

The regulation of secretion in exocrine and endocrine cells has long been of interest. Electron microscopic and other studies have demonstrated that secretory proteins synthesized on ribosomes are transported by the rough ER to the Golgi complex where they are concentrated into secretory granules. During active secretion, secretory granules fuse with the cell membrane, liberating and discharging their contents into the perivascular spaces. When secretory activity is suppressed in anterior pituitary cells, undischarged secretory granules may be degraded by lysosomes. In the parathyroid gland, evidence indicates that the level of blood Ca ions regulates both the production and release of parathormone. Thus, when serum Ca is low, synthesis and release of parathormone are both stimulated; when serum Ca is elevated, these processes are inhibited.

Download Full-text