A conserved strategy to attack collagen: The activator domain in bacterial collagenases unwinds triple-helical collagen.

Bacterial collagenases are important virulence factors, secreted by several pathogenic Clostridium, Bacillus, Spirochaetes, and Vibrio species. Yet, the mechanism by which these enzymes cleave collagen is not well understood. Based on biochemical and mutational studies we reveal that collagenase G (ColG) from Hathewaya histolytica recognizes and processes collagen substrates differently depending on their nature (fibrillar vs. soluble collagen); distinct dynamic interactions between the activator and peptidase domain are required based on the substrate type. Using biochemical and circular dichroism studies, we identify the presumed noncatalytic activator domain as the single-domain triple helicase that unwinds collagen locally, transiently, and reversibly.

Outcomes of collagenase Clostridium histolyticum in men with ventral curvatures: an updated series.

BACKGROUND: The efficacy and safety of collagenase Clostridium histolyticum (CCH) have been demonstrated in the treatment of men with Peyronie's disease (PD); however, the pivotal clinical trials excluded men with ventral penile curvature. AIM: The study sought to evaluate outcomes of CCH treatment in men with ventral curvatures secondary to PD. METHODS: Men with PD treated with CCH were identified from a prospective database. Patients received up to 4 series of CCH injections using a progressively modified protocol over time. Results were compared between those with baseline ventral vs nonventral penile curvatures. OUTCOMES: Changes in penile curvature, Peyronie's Disease Questionnaire scores, International Index of Erectile Function scores, nonstandardized assessments, and adverse events. RESULTS: A total of 560 men with PD (85 ventral curvature, 475 nonventral curvature) were included in the analysis. Baseline median curvature was 60.0° (interquartile range, 48.8°-75.0°) in the ventral cohort and 65.0° (interquartile range, 45.0°-80.0°) in the nonventral cohort. Median change from baseline penile curvature was -25.0° in the ventral cohort vs -24.0° in the nonventral cohort (P = .08, between-group comparison), which corresponded to curvature reductions of 44.7% and 33.6%, respectively (P = .03). In the subset of patients who completed CCH treatment (ie, received 8 injections or discontinued early because of patient satisfaction with curvature reduction), median change from baseline was -35.0° in the ventral cohort vs -25.0° in the nonventral cohort (P < .05); median percent improvement was 48.3% and 37.5%, respectively (P = .11). Median change from baseline in Peyronie's Disease Questionnaire and International Index of Erectile Function domain scores and adverse events were similar between cohorts, with the exception of possibly higher hematoma rates in the nonventral group (50% vs 37%; P = .05). No urethral injuries were sustained in either cohort. CLINICAL IMPLICATIONS: Data support the use of CCH for the treatment of ventral as well as nonventral penile curvatures in men with PD. STRENGTHS AND LIMITATIONS: Study strengths are the inclusion of a general clinical population of men with PD, the prospective design, and the relatively large series of men with ventral curvature. Limitations include the single-center and observational nature of the study. CONCLUSION: CCH was safe and effective in the treatment of both ventral and nonventral penile curvatures in men with PD.

Significant Geographic Variation in Access to Certified Collagenase Clostridium histolyticum Injectors for Peyronie's Disease Throughout the United States.

INTRODUCTION: We characterize the geographic distribution of providers trained to inject Clostridium histolyticum and identify areas with low provider availability. METHODS: We utilized a publicly available search tool to identify clinical sites offering Clostridium histolyticum in the US The data gathered included the provider's name, specialty, address, and whether the site was considered high-volume (ie, administer ≥20 Clostridium histolyticum injections per year). Data were compared to the AUA Census. RESULTS: In total, 2,388 clinical sites offering Clostridium histolyticum were identified. A total of 894 sites (37%) were high-volume sites. The mean number of locations offering Clostridium histolyticum per 100,000 state residents was 0.69 (SD 0.27). Georgia (1.28), Rhode Island (1.13), and Alaska (1.10) had the highest number, whereas New Mexico (0.10), Maine (0.22), and Delaware (0.30) had the lowest. The mean proportion of urologists providing Clostridium histolyticum to total urologists was 0.17 (SD 0.07). The 3 states with the highest proportion were Georgia (0.37), Alaska (0.31), and Utah (0.30), whereas New Mexico (0.03), Maine (0.05), and Vermont (0.06) had the lowest. CONCLUSIONS: States with low numbers of clinical sites offering Clostridium histolyticum per 100,000 residents relative to other states also had a low total ratio of urologists offering Clostridium histolyticum as a treatment. There is room for urologists in these states and others to expand their practice to offer Clostridium histolyticum and improve patient access to this important nonsurgical treatment option.

Comparison of Collagenase Clostridium histolyticum to Surgery for the Management of Peyronie's Disease: A Randomized Trial.

PURPOSE: Since Food and Drug Administration approval of collagenase Clostridium histolyticum for Peyronie's disease, there has been significant debate regarding its role and comparable efficacy to surgery. MATERIALS AND METHODS: A randomized, controlled trial was performed of Peyronie's disease men treated with either collagenase C histolyticum + RestoreX penile traction therapy + sildenafil or penile surgery + RestoreX penile traction therapy + sildenafil, with 3-month data presented. Primary objectives were overall satisfaction, subjective changes in erectile function, penile sensation, penile length, and changes in the International Index of Erectile Function-Erectile Function Domain score. Secondary outcomes included objective changes in length, curve, adverse events, and other standardized and nonstandardized questionnaires. RESULTS: A total of 40 men were enrolled, with 38 (collagenase C histolyticum group = 19, surgery group = 19) completing treatment and having 3-month data available. All demographic and clinicopathological variables were similar between groups. Following treatment, 50% of men in the collagenase C histolyticum group reported being very satisfied (vs 21% in the surgery group, P = .08) and noted better subjective erectile function (100% vs 68%, P = .03) and penile length (88% vs 16%, P < .0001), lesser impacts on penile sensation (75% vs 11% no change, P < .001), and similar International Index of Erectile Function-Erectile Function Domain changes (+1.5 vs +2.5, P = .91). Objectively, men in the surgery group had greater curve improvements (84% vs 54%, P < .01) and higher rates of adverse events (50 vs 13 events, P < .001) but decreased penile length (-0.5 cm vs +1.0 cm, P < .01). CONCLUSIONS: At 3 months posttreatment, collagenase C histolyticum + RestoreX penile traction therapy + sildenafil results in lesser curve improvements but greater penile length and fewer adverse events, including impacts on subjective erectile function and sensation, than men treated with surgery.

Pharmacotherapies in Dupuytren Disease: Current and Novel Strategies.

Dupuytren disease is a benign, progressive fibroproliferative disorder of the hands. To date, only one pharmacotherapy (clostridial collagenase) has been approved for use in Dupuytren disease. There is a great need for additional nonsurgical methods that can be used to either avoid the risks of invasive treatments or help minimize recurrence rates following treatment. A number of nonsurgical modalities have been discussed in the past and continue to appear in discussions among hand surgeons, despite highly variable and often poor or no long-term clinical data. This article reviews many of the pharmacotherapies discussed in the treatment of Dupuytren disease and novel therapies used in inflammation and fibrosis that offer potential treatment options.

Efficacy of collagenase Clostridium histolyticum in men with congenital penile curvature.

BACKGROUND: Collagenase Clostridium histolyticum (CCH) has been proven effective in multiple subpopulations of PD men; however, no studies have evaluated its role with congenital penile curvature (CPC). AIM: To evaluate the safety and efficacy of CCH in men with CPC. METHODS: A prospective registry was queried of men undergoing CCH injections at our institution. Beginning in 2016, CCH was administered to CPC men using a similar protocol to PD. A comparative analysis was performed between cohorts to evaluate the safety and efficacy of therapy. OUTCOMES: Objective measures included penile length and curvature, while subjective outcomes included standardized (International Index of Erectile Function and Peyronie's Disease Questionnaire) and non-standardized assessments. Curvature outcomes were categorized as follows: 1 - included all men, with the most recent assessment considered final, and 2 - only men who had completed eight CCH injections or stopped early due to satisfaction. RESULTS: From 2014 to Oct 2021, a total of 453 men (408 PD, 45 CPC) underwent one or more CCH injections. In comparing cohorts, CPC men were younger (33 vs 58 years, p<0.0001), had lesser baseline curvatures (52.5 vs 65°, p<0.01), more ventral curves (25.7 vs 9.2%, p<0.01), and longer penile lengths (12.5 vs 12.0 cm, p=0.04). Following treatment, both cohorts experienced similar curvature improvements. Specifically, PD men experienced 20-25° or 33-35% improvements depending on definition compared to 25-30° or 40-50% in CPC men (all p-values >0.05). CPC and PD men also demonstrated similar changes on standardized questionnaires, with the exception of the Psychological and Physical subdomain, which was more improved in CPC men (-11 vs -4, p<0.01). Baseline curvature was positively correlated with greater absolute (degree) and relative (percent) improvements. Adverse events were similar between groups after controlling for confounders. CLINICAL IMPLICATIONS: CCH may be safely and effectively administered in men with CPC. These data provide the first evidence for the efficacy of a non-surgical therapeutic option in this cohort. STRENGTHS AND LIMITATIONS: Strengths - large, prospective series with standardized assessments; Limitations - non-randomized study, short-term follow-up, and lack of standardized method to differentiate CPC from PD. CONCLUSIONS: CCH may be safely and effectively administered to men with CPC, with similar success rates compared to PD. Increasing curvature was associated with greater absolute (degree) and relative (percent) improvements, supporting the role for CCH in men with mild, moderate, or severe curvatures. External validation is warranted prior to routine implementation.

Patient Insight With Collagenase Treatment for Dupuytren: A Prospective Study.

PURPOSE: There appears to be controversy regarding differing patient and physician perceptions of adverse effects (AEs) in the treatment of Dupuytren disease with collagenase clostridium histolyticum (CCH). The aim of this study was to compare the number, type, and severity of AEs perceived and reported by patients and by their physician METHODS: To assess AEs following CCH injection in a standardized way, patients were given a list of predefined complications and asked to rate their severity on a 4-point Likert scale ranging from 1 (serious) to 4 (insignificant). RESULTS: Eighty-five patients were included. Patients reported fewer AEs than their physician (mean, 1.48 vs 2.18). There was no agreement between physician- and patient-reported AEs except for skin lacerations, which showed fair agreement (κ = 0.257). CONCLUSIONS: Patients and physicians differ in their evaluation of AEs due to CCH treatment in Dupuytren disease. A fair level of agreement was observed for skin lacerations. CLINICAL RELEVANCE: Greater consensus is needed when defining AEs associated with CCH in the treatment of Dupuytren disease.

Short-term efficacy and adverse effects of collagenase clostridium histolyticum injections, percutaneous needle fasciotomy and limited fasciectomy in the treatment of Dupuytren's contracture: a network meta-analysis of randomized controlled trials.

AIMS: Dupuytren's contracture (DC) is a chronic debilitating fibroproliferative disorder. Common treatment options include collagenase clostridium histolyticum injections (CI), percutaneous needle fasciotomy (NF) and limited fasciectomy (LF). Superiority of one specific treatment remains controversial. This study aims to assess the short-term efficacy and safety of CI, NF, and LF for the treatment of DC. METHODS: We included randomized controlled trials of CI compared with placebo, NF and LF for patients with DC. PubMed, Embase and the Cochrane Library were searched from inception to August 2021. Contracture reduction rates in treated joints (within 0-5° of full extension within 30 days), relative reduction in total passive extension deficit (TPED), occurrence of one or more adverse events and number of treatment-related adverse events per patient were the outcomes of interest. The Cochrane risk-of-bias tool was employed for quality assessment of the studies. A network meta-analysis was performed using MetaXL. RESULTS: Nine studies met our inclusion criteria (n = 903). Overall, risk bias was mixed and mostly low. Short term TPED reduction achieved with LF was superior compared to CI and NF. Although CI achieved greater TPED reduction compared to NF, it was associated with the highest risk of overall adverse effects. The analyzed data was limited to a maximum three-year follow-up period and therefore insufficient for long-term outcome evaluation. CONCLUSIONS: In DC, LF may be able to provide patients with severe disease, superior flexion contracture release postoperatively. CI is a valid treatment alternative to NF, however the higher risk of overall adverse effects must be considered. The quality-of-evidence is limited due to short-term follow-up periods and a lack of standardized definitions of complications and adverse events.

Predictors of Curvature Improvement in Men With Peyronie's Disease Treated With Intralesional Collagenase Clostridium Histolyticum.

INTRODUCTION: Penile curvature is the most common abnormality that is observed by men with Peyronie's disease (PD). Collagenase Clostridium histolyticum (CCH) has become a standard treatment for PD patients. AIM: To identify predictor factors associated with improvements of penile curvature outcomes in men with PD treated with CCH. METHODS: We retrospectively collected the data of patients with PD treated with CCH up to 8 injections divided into 4 cycles between January 2014 and July 2020. Per protocol, penile curvature was assessed at baseline, and after the second and ford CCH cycle. If after cycle 2, curvature demonstrated no improvement, or penile curvature was significantly improved and the patient was happy, no further treatment was recommended. However, if penile curvature was significantly improved and the patient remained dissatisfied, 4 cycles were completed. Three categories of response were evaluated: improvement (≥10 degrees or ≥20%, either 1 happens), unchanged (±10 degrees or ±20%) or worsened (≥10 degrees or ≥20%, either 1 happens). Logistic regression analyses were performed to evaluate predictive factors associated with penile curvature improvements. OUTCOMES: Degrees of the curvature changes between the baseline and after the cycles of CCH. RESULTS: A total of 114 patients underwent CCH treatment. Median age was 57 years. Median PD duration was 11 months. At baseline, mean curvature was 47 degrees, 65% had dorsal curvature, 53% mid-shaft location, and 15% calcification. After CCH treatment, the mean final curvature was 40 degrees. A total of 44% improved the curvature, 39% had no change while 17% worsened after CCH treatment. Of men who had penile curvature improvement with CCH treatment, the mean curvature decreasing in degrees and percentage were 22 degrees and 41%, respectively. Men with baseline curvature ≤ 30, 31-59, and ≥ 60 degrees, the percentage curvature improvement were 29%, 43%, and 60%, respectively. Baseline curvature was the only significant predictor of penile curvature improvement after CCH (OR 1.33, 95% CI = 1.1, 1.7). CLINICAL IMPLICATIONS: We confirmed baseline penile curvature is the most important predictive factor, and this is the first report describing proportions of penile curvature improvement with CCH treatment. STRENGTHS AND LIMITATIONS: This study has several strengths, including the use of validated instruments. Nonetheless, there are limitations: the retrospective nature of the study, a single institution; and modelling device was not controlled. CONCLUSION: Penile curvature improvement was significantly more common in patients with greater baseline curvature, reaching up to 60% for patients with ≥ 60 degrees. Flores JM, Nascimento B, Punjani N, et al. Predictors of Curvature Improvement in Men With Peyronie's Disease Treated With Intralesional Collagenase Clostridium Histolyticum. J Sex Med 2022;19:1680-1686.

Discovery and Characterization of Synthesized and FDA-Approved Inhibitors of Clostridial and Bacillary Collagenases.

In view of the worldwide antimicrobial resistance (AMR) threat, new bacterial targets and anti-infective agents are needed. Since important roles in bacterial pathogenesis have been demonstrated for the collagenase H and G (ColH and ColG) from Clostridium histolyticum, collagenase Q1 and A (ColQ1 and ColA) from Bacillus cereus represent attractive antivirulence targets. Furthermore, repurposing FDA-approved drugs may assist to tackle the AMR crisis and was addressed in this work. Here, we report on the discovery of two potent and chemically stable bacterial collagenase inhibitors: synthesized and FDA-approved diphosphonates and hydroxamates. Both classes showed high in vitro activity against the clostridial and bacillary collagenases. The potent diphosphonates reduced B. cereus-mediated detachment and death of cells and Galleria mellonella larvae. The hydroxamates were also tested in a similar manner; they did not have an effect in infection models. This might be due to their fast binding kinetics to bacterial collagenases.

The Effect of Anticoagulation on the Treatment of Dupuytren Contracture with Collagenase.

BACKGROUND: There have been no formal studies of the use of collagenase clostridium histolyticum (Xiaflex; Endo Pharmaceuticals, Malvern, Pa.) in the anticoagulated patient. Previous study on its use excluded patients receiving anticoagulation therapy. This study reviewed the effects of anticoagulation use in patients undergoing collagenase clostridium histolyticum injection and cord rupture to determine safety and efficacy. METHODS: The authors retrospectively reviewed patients receiving collagenase clostridium histolyticum injections for Dupuytren contracture between 2010 and 2017. Outcomes included type of anticoagulation, skin tear, tendon rupture, lymphadenopathy, sensory abnormality, hematoma, and bleeding. RESULTS: A total of 388 injections were performed in 197 patients. The average clinical follow-up was 311 days. Fifty-two percent of patients (n = 201) were anticoagulated. The vast majority were taking aspirin (acetylsalicylic acid) (n = 182), followed by warfarin (n = 27), clopidogrel (n = 9), apixaban (n = 8), rivaroxaban (n = 4), and dabigatran (n = 2). There were 42 skin tears, 21 in the anticoagulated group. One tendon rupture occurred in the anticoagulated group (acetylsalicylic acid) and none occurred in the nonanticoagulated group (p = 0.34). No patients experienced sensory abnormalities, a hematoma requiring intervention, or uncontrollable bleeding. CONCLUSIONS: Anticoagulation is commonly encountered by hand surgeons treating Dupuytren contracture. There is no significant difference in complications in the anticoagulated patient versus the nonanticoagulated patient. It is safe to perform collagenase clostridium histolyticum injections for Dupuytren contracture in the anticoagulated patient, regardless of the type of anticoagulation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

The collagenase of the bacterium Clostridium histolyticum does not favor metastasis of breast cancer.

BACKGROUND: Breast cancer is the most common malignancy among women worldwide. As survival rates increase, breast reconstruction and quality of life gain importance. Of all women undergoing breast reconstruction, approximately, 70% opt for silicone implants and 50% of those develop capsular contracture, the most prevalent long-term complication. The collagenase of the bacterium Clostridium histolyticum (CCH) showed promising results in the therapy of capsule contracture; however, its influence on residual cancer cells is unknown. The aim of this study was to investigate whether CCH-treatment negatively impacts breast cancer cells in vitro and in vivo. METHODS: MDA-MB-231 and MCF-7 cells were used in this study. In vitro, we tested the influence of CCH on proliferation, wound healing, migration and cell cycle by MTT-assay, scratch-assay, transwell-migration-assay, and flow cytometry. In vivo, solid tumors were induced in immune-deficient mice. CCH was injected into the tumors and tumor growth and metastasis formation was monitored by caliper measurement, in vivo bioluminescence imaging and histology. Gene expression analysis was performed by microarray including 27,190 genes. RESULTS: CCH-incubation led to a dose-dependent reduction in proliferation for both cell lines, while wound healing was reduced only in MDA-MB-231 cells. No morphological alterations were monitored in cell cycle or apoptosis. In vivo, bioluminescence imaging and histology did not show any evidence of metastasis. Although CCH led to changes in gene expression of breast cancer cells, no relevant alterations in metastasis-related genes were monitored. CONCLUSION: CCH has no impact on tumor growth or metastasis formation in vitro and in vivo. This paves the way for first clinical trials.

Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications.

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.

Limited Fasciectomy Versus Collagenase Clostridium histolyticum for Dupuytren Contracture: A Propensity Score Matched Study of Single Digit Treatment With Minimum 5 Years of Telephone Follow-Up.

PURPOSE: The purpose of this study was to compare reintervention and perceived recurrence, with minimum 5 years of telephone follow-up, after limited fasciectomy or collagenase Clostridium histolyticum (CCH) in the treatment of Dupuytren contracture affecting a single digit. METHODS: We performed a retrospective cohort study of 48 patients with single digit treatment who underwent limited surgical fasciectomy at one hospital and 111 patients who underwent CCH treatment at a second hospital from 2010 to 2013. Patients were contacted by telephone about reintervention and perceived recurrence. Average length of telephone follow-up was 7.3 years in the CCH group and 7.4 years in the surgery group. The 2 groups were compared using 2 methods to control for potential confounding bias: (1) propensity score matching and (2) multivariable analysis accounting for potential confounders. RESULTS: After propensity score matching, there were 44 patients in each group with similar disease and demographic characteristics. Rates of reintervention and perceived recurrence were significantly higher in the CCH group than the surgery group at a minimum of 5 years following treatment. CONCLUSIONS: Long-term overall reintervention and perceived recurrence following treatment of Dupuytren contracture affecting a single digit were higher with CCH treatment than surgical fasciectomy when comparing groups with similar baseline characteristics. Our findings may be used to counsel patients on the durability of the outcomes of treatment when considering treatment options for Dupuytren contractures. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Continuing Collagenase Clostridium Histolyticum Injections Among Initial Nonresponders Results in Significant Curvature Improvements in the Majority of Peyronie's Disease Men.

BACKGROUND: It is currently unclear if men with Peyronie's Disease (PD) who achieve minimal benefits with the first 2 series of Collagenase Clostridium Histolyticum (CCH) injections should continue with additional injections. AIM: To analyze curvature improvements from the final two series of CCH injections based on amount of improvement during the first 2 series. METHODS: A prospective registry was analyzed of all men undergoing CCH injections for PD at a single institution. Men were included if they had completed a full 4 series (8 injections) of CCH and had baseline, interval (after 2 series), and/or final (after 4 series) curvature assessments available. Men were stratified into cohorts using baseline-to-interval assessments of ≤10° (or ≤20%) and >10° (or >20%), and improvements were compared using interval-to-final assessments. OUTCOMES: The primary outcome was interval-to-final curvature improvements stratified by ≤10°/>10° or ≤20%/>20% improvements achieved during the baseline-to-interval period. Secondary outcomes included analyses of demographic and pathophysiologic variables to determine associations with significant improvements during the final 2 CCH series. RESULTS: A total of 296 PD men were identified as receiving at least one CCH injection, of whom 175 had baseline-to-interval, 84 interval-to-final, and 115 with baseline-to-final measurements. Mean age was 56.6, PD duration 28.6 months, baseline curvature 63.4°, hourglass deformity 36.2%, and calcification 20%. Mean overall curve improvement was -21.5° (33.1%). Among men who experienced ≤20% improvements after 2 series, the mean subsequent curvature change was -24.6% during the final two series (vs +4.3% of those with >20% initial improvement, P< .001), and they were 2.7x more likely to experience >20% subsequent curve improvements. Thirty-one percent of those who achieved >10° during the first 2 series experienced benefits during the final 2 series compared to 70% of men who had ≤10° improvement initially. No demographic or pathophysiological variables predicted likelihood for improvements during the final 2 series of injections. CLINICAL IMPLICATIONS: Men who fail to achieve significant benefits with 2 series of CCH injections may benefit from completing the final 2 series. STRENGTHS AND LIMITATIONS: Strengths including a relatively large, prospective series. Limitations include a single center, nonrandomization, nonblinded assessments, and restriction to men who completed eight injections. CONCLUSIONS: In the current series, approximately 2/3 of men who fail to achieve >10° or 20% curve improvements with an initial 2 series of CCH injections achieved >10° or 20% improvements with the subsequent 2 series. Alom M, Burgon H, Ziegelmann M, et al. Continuing Collagenase Clostridium Histolyticum Injections Among Initial Nonresponders Results in Significant Curvature Improvements in the Majority of Peyronie's Disease Men. J Sex Med 2021;18:1092-1098.

Glucokinase Inactivation Paradoxically Ameliorates Glucose Intolerance by Increasing ß-Cell Mass in db/db Mice.

Efficacy of glucokinase activation on glycemic control is limited to a short-term period. One reason might be related to excess glucose signaling by glucokinase activation toward ß-cells. In this study, we investigated the effect of glucokinase haploinsufficiency on glucose tolerance as well as ß-cell function and mass using a mouse model of type 2 diabetes. Our results showed that in db/db mice with glucokinase haploinsufficiency, glucose tolerance was ameliorated by augmented insulin secretion associated with the increase in ß-cell mass when compared with db/db mice. Gene expression profiling and immunohistochemical and metabolomic analyses revealed that glucokinase haploinsufficiency in the islets of db/db mice was associated with lower expression of stress-related genes, greater expression of transcription factors involved in the maintenance and maturation of ß-cell function, less mitochondrial damage, and a superior metabolic pattern. These effects of glucokinase haploinsufficiency could preserve ß-cell mass under diabetic conditions. These findings verified our hypothesis that optimizing excess glucose signaling in ß-cells by inhibiting glucokinase could prevent ß-cell insufficiency, leading to improving glucose tolerance in diabetes status by preserving ß-cell mass. Therefore, glucokinase inactivation in ß-cells, paradoxically, could be a potential strategy for the treatment of type 2 diabetes.

Biochemical characterisation of a collagenase from Bacillus cereus strain Q1.

Collagen is the most abundant protein in higher animals and as such it is a valuable source of amino acids and carbon for saprophytic bacteria. Due to its unique amino acid composition and triple-helical tertiary structure it can however only be cleaved by specialized proteases like the collagenases secreted by some bacteria. Among the best described bacterial collagenases are ColG and ColH from Clostridium histolyticum. Many Bacillus species contain homologues of clostridial collagenases, which play a role in some infections caused by B. cereus. Detailed biochemical and enzymatic characterizations of bacillial collagenases are however lacking at this time. In an effort to close this gap in knowledge we expressed ColQ1 from B. cereus strain Q1 recombinantly, investigated its metal dependency and performed peptide, gelatin and collagen degradation assays. Our results show that ColQ1 is a true collagenase, cleaving natively folded collagen six times more efficiently than ColG while at the same time being a similarly effective peptidase as ColH. In both ColQ1 and ColG the rate-limiting step in collagenolysis is the unwinding of the triple-helix. The data suggest an orchestrated multi-domain mechanism for efficient helicase activity.