Diabetes is one of the major challenges of modern medicine, as it is considered a global epidemic of the XXI century. The disease often leads to the development of serious, health threatening complications. Diabetic foot syndrome is a characteristic set of anatomical and molecular changes. At the macroscopic level, major symptoms are neuropathy, ischemia and chronic ulceration of the lower limb. In every third patient, the neuropathy develops into Charcot neuroarthropathy characterized by bone and joints deformation. Interestingly, all these complications are a result of impaired healing processes and are characteristic for diabetes. The specificity of these symptoms comes from impaired molecular mechanisms observed in type 1 and type 2 diabetes. Decreased wound and fracture healing reflect gene expression, cellular response, cell functioning and general metabolism. Here we present a comprehensive literature update on the molecular factors contributing to diabetic foot syndrome.

In bacteria, the so-called stringent response is responsible for adaptation to changing environmental conditions. This response is mediated by guanosine derivatives [(p)ppGpp], synthesized by either large mono-functional RelA or bi-functional SpoT (synthesis and hydrolysis) enzymes in β - and γ - proteobacteria, such as Escherichia coli . In Firmicutes and α - , δ - , and 𝜀 - proteobacteria, large bifunctional Rel-SpoT-homologs (RSH), often accompanied by small (p)ppGpp synthetases and/or hydrolases devoid of regulatory domains, are responsible for (p)ppGpp turnover. Here, we report on surprising in vitro and in vivo properties of an RSH enzyme from Methylobacterium extorquens (RSH Mex ). We find that this enzyme possesses some unique features, e.g., it requires cobalt cations for the most efficient (p)ppGpp synthesis, in contrast to all other known specific (p)ppGpp synthetases that require Mg 2+ . In addition, it can synthesize pppApp, which has not been demonstrated in vitro for any Rel/SpoT/RSH enzyme so far. In vivo , our studies also show that RSH Mex is active in Escherichia coli cells, as it can complement E. coli ppGpp 0 growth defects and affects rrnB P1- lacZ fusion activity in a way expected for an RSH enzyme. These studies also led us to discover pppApp synthesis in wild type E. coli cells (not carrying the RSH Mex enzyme), which to our knowledge has not been demonstrated ever before. In the light of our recent discovery that pppApp directly regulates E. coli RNAP transcription in vitro in a manner opposite to (p)ppGpp, this leads to a possibility that pppApp is a new member of the nucleotide second-messenger family that is widely present in bacterial species.

Precise regulation of gene expression is crucial for bacteria to respond to changing environmental conditions. In addition to protein factors affecting RNA polymerase (RNAP) activity, second messengers play an important role in transcription regulation, such as well-known effectors of the stringent response: guanosine 5'triphosphate-3'diphosphate and guanosine 3', 5'-bis(diphosphate) [(p)ppGpp]. Although much is known about importance of the 5' and 3' moieties of (p)ppGpp, the role of the guanine base remains somewhat cryptic. Here, we use (p)ppGpp's adenine analogs [(p)ppApp] to investigate how the nucleobase contributes to determine its binding site and transcriptional regulation. We determined X-ray crystal structure of Escherichia coli RNAP-(p)ppApp complex, which shows the analogs bind near the active site and switch regions of RNAP. We have also explored the regulatory effects of (p)ppApp on transcription initiating from the well-studied E. coli rrnB P1 promoter to assess and compare properties of (p)ppApp with (p)ppGpp. We demonstrate that contrary to (p)ppGpp, (p)ppApp activates transcription at this promoter and DksA hinders this effect. Moreover, pppApp exerts a stronger effect than ppApp. We also show that when ppGpp and pppApp are present together, the outcome depends on which one of them was pre-incubated with RNAP first. This behavior suggests a surprising Yin-Yang like reciprocal plasticity of RNAP responses at a single promoter, occasioned simply by pre-exposure to one or the other nucleotide. Our observations underscore the importance of the (p)ppNpp's purine nucleobase for interactions with RNAP, which may lead to a better fundamental understanding of (p)ppGpp regulation of RNAP activity.

Charcot arthropathy is one of the most serious complications of diabetic foot syndrome that leads to amputation of the affected limb. Since there is no cure for Charcot arthropathy, early diagnosis and implementation preventive care are the best available treatment. However, diagnosis is hindered by obscure clinical picture of the disease and lack of molecular markers for its early detection. Results of recent research suggest that OPG-RANKL-RANK axis regulating bone metabolism can be associated with Charcot arthropathy and that SNPs in OPG gene are associated with the disease. Here we report the results of comprehensive analysis of ten SNPs in OPG, RANKL and RANK genes in 260 subjects divided into diabetes, neuropathy and Charcot arthropathy groups. Besides genotype analysis we performed linkage disequilibrium and hierarchical clustering to obtain information about correlation between SNPs. Our results show that OPG 245T/G (rs3134069) and OPG 1217C/T (rs3102734) polymorphisms co-occur in patients with Charcot arthropathy (r2 = 0.99). Moreover, hierarchical clustering revealed a characteristic profile of all SNPs in Charcot arthropathy and neuropathy, which is distinct from control group. Our results suggest that analysis of multiple SNPs can be used as potential marker of Charcot arthropathy and provide insight into possible molecular mechanisms of its development.

The Mesh1 class of hydrolases found in bacteria, metazoans and humans was discovered as able to cleave an intact pyrophosphate residue esterified on the 3 hydroxyl of (p)ppGpp in a Mn 2+ dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from Drosophila melanogaster and human MESH1 also extends to the (p)ppApp purine analogs. More importantly, we developed real time enzymatic assays, coupling ppNpp hydrolysis to NADH oxidation and pppNpp hydrolysis to NADP + reduction, which facilitate estimation of kinetic constants. Furthermore, by using this assay technique we confirmed TLC observations and also revealed that purified small alarmone hydrolase (SAH Mex) from Methylobacterium extorquens displays a strong hydrolase activity toward (p)ppApp but only negligible activity toward (p)ppGpp. In contrast, the substrate specificity of the hydrolase present in catalytically active N-terminal domain of the RSH protein from Streptococcus equisimilis (Rel Seq) includes (p)ppGpp but not (p)ppApp. It is noteworthy that the RSH protein from M. extorquens (RSH Mex) has been recently shown to synthesize both (p)ppApp and (p)ppGpp.