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Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.
Objective: The pathophysiological mechanisms underlying chronic pancreatitis (CP) are still poorly understood. Human cationic (TRY1) and anionic (TRY2) trypsins are the two major trypsin isoforms and their activities are tightly regulated within pancreatic acinar cells. Typically, they exist in a molar ratio of 2:1 (cationic:anionic). This ratio is reversed during chronic alcohol abuse, pancreatic cancer, or pancreatitis due to selectively upregulated expression of TRY2, causing anionic trypsin to become the predominant isoform. The involvement of TRY2 in pancreatitis is considered limited due to the absence of disease-causing mutations and its increased prevalence for autoproteolysis. However, exacerbated pancreatitis in TRY2 overexpressing mice was recently demonstrated. Here, we aim to elucidate the molecular structure of human anionic trypsin and obtain insights into the autoproteolytic regulation of tryptic activity.
Methods: Trypsin isoforms were recombinantly expressed in E. coli, purified and refolded. Enzymatic activities of all trypsin isoforms were determined and crystals of TRY2 were grown using the vapor-diffusion method. The structure was solved by molecular replacement and refined to a resolution of 1.7 Å. Equilibration molecular dynamics simulations were used to generate the corresponding TRY1–TRY1 model.
Results: All trypsin isoforms display similar kinetic properties. The crystal structure of TRY2 reveals that the enzyme crystallized in the autoproteolytic state with Arg122 placed in the S1 binding pocket and the corresponding loop cleaved. The TRY2–TRY2 dimer confirms a previously hypothesized autoinhibitory state with an unexpectedly large binding interface.
Conclusion: We provide a structure of TRY2, which is the predominant trypsin isoform in chronic pancreatitis and pancreatic cancer. A proposed autoinhibition mode was confirmed and the structural basis of the autoproteolytic failsafe mechanism elucidated.
Pancreatitis is an inflammatory disorder of the pancreas with a mortality rate of 5% and severe negative effects on the quality of life. Of all non-malignant gastrointestinal diseases, it is the most common reason for hospitalization. Pancreatitis is a disease of multiple etiologies with different underlying pathomechanisms. Due to the diversity of mechanisms by which homeostasis within the exocrine pancreas can be disrupted, finding appropriate therapeutic approaches is challenging. Current treatment options are inadequate and are mostly limited to supportive treatment like fluid administration, bowel rest, antibiotics and pain control. Although significant advancements have been achieved in recent decades, the mortality rate for pancreatitis has not decreased. Furthermore, progress is slow due to limited patient sample availability and lack of an appropriate cell model. Taking samples from a human pancreas is typically avoided, because damaging the pancreatic tissue can itself induce pancreatitis. Additionally, while it is possible to keep individual acini in culture, it is not possible to grow pancreatic acinar cells. Thus, less appropriate cell models, often derived from pancreatic cancer samples, have to be used. The most common animal model for pancreatitis is mice, with caerulein administration being the most common method of inducing pancreatitis. However, the use of animal models has significant drawbacks, as they are time-consuming, costly, and pose ethical questions. Furthermore, exposing the pancreas to appropriate stimuli in animal models is difficult. For example, alcohol is the leading cause of pancreatitis in humans, but is typically avoided by animals. Thus, alcohol feeding methods had to be developed to overcome the natural aversion of rodents to alcohol. Results obtained from animal models are also often not transferable into clinical trials and outcomes in humans remain largely unpredictable. Due to the lack of experimental models, our understanding of this highly complex disease is still limited and significant progress is required for the development of effective therapy options.
In this dissertation recombinantly expressed trypsin isoforms and variants of the serine protease inhibitor Kazal-type 1 (SPINK1) inhibitor are used to investigate mechanisms, by which tryptic activity is regulated in pancreatic acinar cells. With premature tryptic activity in the exocrine pancreas being the common focal point of most etiologies connected to pancreatitis, trypsin represents by far the most promising target for treating pancreatitis. Understanding the mechanisms by which the pancreas protects itself and rationalizing mutations that can undermine these protective mechanisms, are important steps towards developing effective therapies.
(1) The serine protease inhibitor Kazal type 1 (SPINK1) inhibits trypsin activity in zymogen granules of pancreatic acinar cells. Several mutations in the SPINK1 gene are associated with acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). The most common variant is SPINK1 p.N34S. Although this mutation was identified two decades ago, the mechanism of action has remained elusive. (2) SPINK1 and human cationic trypsin (TRY1) were expressed in E. coli, and inhibitory activities were determined. Crystals of SPINK1–TRY1 complexes were grown by using the hanging-drop method, and phases were solved by molecular replacement. (3) Both SPINK1 variants show similar inhibitory behavior toward TRY1. The crystal structures are almost identical, with minor differences in the mutated loop. Both complexes show an unexpected rotamer conformation of the His63 residue in TRY1, which is a member of the catalytic triad. (4) The SPINK1 p.N34S mutation does not affect the inhibitory behavior or the overall structure of the protein. Therefore, the pathophysiological mechanism of action of the p.N34S variant cannot be explained mechanistically or structurally at the protein level. The observed histidine conformation is part of a mechanism for SPINK1 that can explain the exceptional proteolytic stability of this inhibitor.
Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.
One of the most common mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene is the N34S variant which is strongly associated with chronic pancreatitis. Although it is assumed that N34S mutation constitutes a high-risk factor, the underlying pathologic mechanism is still unknown. In the present study, we investigated the impact of physiological stress factors on SPINK1 protein structure and trypsin inhibitor function using biophysical methods. Our circular dichroism spectroscopy data revealed differences in the secondary structure of SPINK1 and N34S mutant suggesting protein structural changes induced by the mutation as an impairment that could be disease-relevant. We further confirmed that both SPINK1 (KD of 0.15 ± 0.06 nM) and its N34S variant (KD of 0.08 ± 0.02 nM) have similar binding affinity and inhibitory effect towards trypsin as shown by surface plasmon resonance and trypsin inhibition assay studies, respectively. We found that stress conditions such as altered ion concentrations (i.e. potassium, calcium), temperature shifts, as well as environmental pH lead to insignificant differences in trypsin inhibition between SPINK1 and N34S mutant. However, we have shown that the environmental pH induces structural changes in both SPINK1 constructs in a different manner. Our findings suggest protein structural changes in the N34S variant as an impairment of SPINK1 and environmental pH shift as a trigger that could play a role in disease progression of pancreatitis.