Exploring Protein Space: From Hydrolase to Ligase by Substitution.

The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence-function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one.

Superiomedial Pedicle Breast Reduction for Gigantic Breast Hypertrophy: Experience in 341 Breasts and Suggested Safety Modifications.

BACKGROUND: Reduction mammoplasty in patients with gigantic breast hypertrophy runs a high risk of complication. Traditionally, inferior pedicle reductions or free nipple grafting techniques have been recommended for gigantic breasts on the basis of measurements and expected resection weights. The superiomedial pedicle (SMP) technique has been less commonly used, due to concerns of vascular inadequacy. This study examines the outcomes of SMP in large reductions and outlines suggested modifications for enhanced safety. METHODS: This is a retrospective review of all patients who underwent SMP breast reduction in our institution between 2005 and 2016. Included are cases with resection weights greater than 800 g. RESULTS: A total of 173 patients with 341 breasts were included. Mean sternal notch to nipple (SNN) distance was mean 35.0 ± 6.6 cm (range 23-44.5) on the left and 34.9 ± 6.6 cm (range 18-46) on the right. Mean resection weight was 1152.2 ± 368.6 g (range 810-2926) on the left and 1159.4 ± 326.6 g (range 800-2528) on the right. The total complication rate was 22.7%. Minor complications occurred in 63 (18.6%) breasts. Major complications occurred in 12 (4.1%) breasts. NAC congestion and partial necrosis occurred in 1.8% and total NAC necrosis in 0.9%. CONCLUSION: The SMP reduction technique is a safe option for gigantic breast reduction with comparable complication rates to other techniques. Preoperative measurements or resection weights are not reliable risk factors alone. High tissue density may be a significant risk factor. High-risk breasts mandate surgical planning and should be tailored to include technical modifications as described. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

The Evolution of the Free Fibula Flap for Head and Neck Reconstruction: 21 Years of Experience with 128 Flaps.

BACKGROUND: The free fibula flap is commonly referred to as a "workhorse" for head and neck reconstruction. During our 21-year experience with this flap, we have performed several changes in preoperative planning, operative technique, and postoperative follow-up. PATIENTS AND METHODS: A retrospective cohort study designed to analyze the cohort of patients who underwent free fibula transfer for head and neck reconstruction. Demographics, medical background, operative data, and postoperative outcome were collected. The changes we performed in preoperative planning, operative technique, and postoperative follow-up were assessed and their impact on outcome discussed. RESULTS: During 1998 to 2019 a total of 128 free fibula flaps were transferred for head and neck reconstruction. When comparing the patients treated in the early years to those who were treated in recent years we found no statistically significant difference in minor or major nonmicrosurgical complications in the recipient and donor site and in the rate of take backs due to microsurgical reasons. However total flap failure rate improved from 28% in early years to 8% in recent years (p = 0.012). CONCLUSION: During this 21-year period, we performed several changes in our practice. This included the use of a three-dimensional (3D) prefabricated model of the mandible, a shift toward side-table osteotomies, increasing the rate of osteofascial flaps in contrast to osteocutaneous flaps and the use of an implantable Doppler. These changes, together with a learning curve of the surgical team, significantly improved our overall success rates.

Multiple layers of regulation determine the cellular levels of the Pup ligase PafA in Mycobacterium smegmatis.

Despite being a destructive process, regulated protein degradation is fundamental for proper cell function. While regulated proteolysis in eukaryotes largely involves the ubiquitin-proteasome system (UPS), most bacterial species rely on multiple ATP-dependent proteases, such as the Clp proteases. Mycobacteria and related actinobacterial species also possess a degradation system analogous in its function to the UPS. In this system, a prokaryotic ubiquitin-like protein (Pup) is conjugated to proteins, thereby marking them for proteasomal degradation. A single ligase, PafA, is responsible for Pup conjugation to many protein targets, thus playing a central role in the Pup-proteasome system (PPS). In Mycobacterium smegmatis, a model mycobacterial organism where the PPS is essential under starvation conditions, cellular PafA levels change in response to nutrient availability. Indeed, increased PafA levels are observed upon nutrient limitation. We found that a multi-layered network involving transcriptional, translational and post-translational regulation determines cellular PafA levels. Induced expression is observed at stationary phase, whereas PafA degradation by the proteasome and ClpCP occurs in exponentially growing cells, as opposed to starved cells. In both growth stages, translation attenuation maintains low PafA expression levels. Altogether, these mechanisms establish the dynamics in PafA levels during bacterial growth.

[FACIAL NERVE PARALYSIS - THERAPEUTIC APPROACH, FACIAL REANIMATION AND ADJUNCTIVE TREATMENT].

INTRODUCTION: Paralysis of the facial mimetic muscles causes loss of voluntary and non-voluntary muscle function, as well as facial tone. This is a devastating condition with profound functional, aesthetic and psychological consequences. Etiologies include congenital paralysis and acquired paralysis following viral infection, trauma, head and neck tumors, iatrogenic damage and more. Clinical presentation includes ocular symptoms (dry eye, epiphora, corneal irritation), nasal symptoms (nasal obstruction) and oral symptoms (drooling and speech disturbances). Reconstruction of facial nerve function is based on renewing the neural input to the paralyzed face in parallel with transferring a functioning muscle. The gold standard in long term facial paralysis reanimation includes a two-stage procedure that involves cross-face nerve grafting and later on a free gracilis muscle transfer. This method allows reconstruction of a symmetric, spontaneous and voluntary smile. In cases when cross-face nerve grafting is impossible, a free-gracilis muscle transfer is performed with neural coaptation to another cranial nerve, most commonly the motor nerve to the masseter muscle (of the trigeminal nerve). Non-microsurgical methods for facial reanimation exist, however, nowadays they are rarely performed. In addition to the surgical reconstruction, other surgical and non-surgical procedures are performed for functional and aesthetic symmetrization purposes. These include fat injection to the face, botulinum toxin injection, oculoplastic procedures and more. In this article we describe our patient population with facial nerve paralysis, common facial reanimation procedures, considerations in choosing the appropriate reconstruction procedure and the general approach for treatment of facial paralysis in our multidisciplinary facial paralysis clinic.

Pup-Click-A New Chemoenzymatic Method for the Generation of Singly Pupylated Targets.

Conjugation of the prokaryotic ubiquitin-like protein (Pup) to cellular proteins tags these proteins for degradation by a proteasome in actinobacteria. To study the Pup-proteasome system in in vitro biochemical assays, Pup-tagged (i.e., pupylated) proteins are often used. However, the purification of a homogeneous preparation of pupylated proteins often suffers from poor yields and limitations in terms of selecting the target protein and its site of pupylation. Here, we report on the development of a biochemical methodology we term Pup-Click for the generation of pupylated protein mimics in vitro. Pup-Click relies on a natural pupylation reaction combined with the use of a synthetic peptide and genetic code expansion via the use of unnatural amino acids and Click chemistry. In principle, this approach allows for conjugation of Pup to any selected target at potentially any desired position. Importantly, pupylated protein mimics generated by Pup-Click are recognized and processed by enzymes of the Pup-proteasome system. As such, Pup-Click can serve as a powerful tool for studying this protein degradation pathway.

Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system.

The proper functioning of any biological system depends on the coordinated activity of its components. Regulation at the genetic level is, in many cases, effective in determining the cellular levels of system components. However, in cases where regulation at the genetic level is insufficient for attaining harmonic system function, posttranslational regulatory mechanisms are often used. Here, we uncover posttranslational regulatory mechanisms in the prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS), the bacterial equivalent of the eukaryotic ubiquitin-proteasome system. Pup, a ubiquitin analog, is conjugated to proteins through the activities of two enzymes, Dop (deamidase of Pup) and PafA (proteasome accessory factor A), the Pup ligase. As Dop also catalyzes depupylation, it was unclear how PPS function could be maintained without Dop and PafA canceling the activity of the other, and how the two activities of Dop are balanced. We report that tight Pup binding and the limited degree of Dop interaction with high-molecular-weight pupylated proteins results in preferred Pup deamidation over protein depupylation by this enzyme. Under starvation conditions, when accelerated protein pupylation is required, this bias is intensified by depletion of free Dop molecules, thereby minimizing the chance of depupylation. We also find that, in contrast to Dop, PafA presents a distinct preference for high-molecular-weight protein substrates. As such, PafA and Dop act in concert, rather than canceling each other's activity, to generate a high-molecular-weight pupylome. This bias in pupylome molecular weight distribution is consistent with the proposed nutritional role of the PPS under starvation conditions.

Balsa wood for precise intra-operative bone contouring in fibula free-flap mandible reconstruction.

OBJECTIVE: To report a simple and novel method for intra-operative planning of fibula free flap reconstruction by means of a balsa wood (BW) model. STUDY DESIGN: Retrospective chart review. METHODS: Between 2010 and 2015, 29 patients underwent mandibular reconstruction by a BW osteotomy design in which a single BW beam (US$4) is cut into segments to match the plate. The segments are then assembled together in a three-dimensional (3D) fashion to conform to the contour of the defect and the angles of attachment. Osteotomies are then performed according to the BW segment lengths and angles. Outcomes were retrospectively analyzed for number of procedures, operative times, and complications between the balsa wood method and more standard techniques, e.g., conventional 3D models. RESULTS: The length of the average mandibular defect was 9.62 cm (4-19), and the mean number of fibula segments was 2.03 ± 0.92 (range 1-4). Only one case (3.4%) necessitated revision surgery. Three patients (10.3%) had minor complications. Comparison of the results of the 13 patients reconstructed solely by BW to the 16 reconstructed by both BW and a 3D-printed model revealed that the use of BW alone did not significantly alter the average number of segments [2.31 (BW) vs 1.69 (combined); P = 0.07] or ischemia time (173 min vs 171 min, respectively, P = 0.938). CONCLUSION: The use of balsa wood as a model for intra-operative planning of fibula free flap osteotomies is an effective, inexpensive, and safe technique.

Breast Reduction and Mastopexy for Repair of Asymmetry After Breast Conservation Therapy: Lessons Learned.

BACKGROUND: Breast conservation therapy (BCT) can cause breast distortion and asymmetry. Repair of this asymmetry by means of breast reduction or mastopexy procedures can be challenging and harbor considerably high rates of complications. METHODS: In this retrospective study, we describe our experience in repairing post-BCT breast asymmetry by performing breast reduction or mastopexy. The surgical protocol we followed consisted of stringent patient selection, thorough surgical planning, basic surgical refinements, and patient education for enhancing the likelihood of achieving a good outcome with minimal surgical complications. RESULTS: Our search of the departmental database identified 25 patients with breast asymmetry who had undergone breast reduction or mastopexy between 2009 and 2017. Corrective surgery was performed 4 years on average after the completion of radiotherapy, and those patients included eleven who had undergone breast reduction and fourteen who had undergone mastopexy on the radiated side. Two patients (8%) had major complications that required further surgery (major fat necrosis, wound infection, and breast deformation), and five patients (20%) had minor complications (infection, minor fat necrosis, wound dehiscence, and nipple congestion). All complications developed on the radiated breast. There was no correlation between the occurrence of complications and patients' demographics, tumor type, tumor location, and breast tissue resection (p > 0.05). CONCLUSION: Only two of our 25 patients had major complications following breast reduction and mastopexy for the repair of asymmetry post-BCT. Following our four-step protocol was instrumental in leading to the successful performance of these procedures. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

How to control an intracellular proteolytic system: Coordinated regulatory switches in the mycobacterial Pup-proteasome system.

Intracellular proteolysis is critical for the proper functioning of all cells, owing to its involvement in a wide range of processes. Because of the destructive nature of protein degradation, intracellular proteolysis is restricted by control mechanisms at almost every step of the proteolytic process. Understanding the coordination of such mechanisms is a challenging task, especially in systems as complex as the eukaryotic ubiquitin-proteasome system (UPS). In comparison, the bacterial analog of the UPS, the Pup-proteasome system (PPS) is much simpler and, therefore, allows for insight into the control of a proteolytic system. This review integrates available information to present a coherent picture of what is known of PPS regulatory switches and describes how these switches act in concert to enforce regulation at the system level. Finally, open questions regarding PPS regulation are discussed, providing readers with a sense of what lies ahead in the field.

Survival of mycobacteria depends on proteasome-mediated amino acid recycling under nutrient limitation.

Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S-proteasome, bacterial prokaryotic ubiquitin-like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup-proteasome system (PPS) is important for virulence, yet its physiological role in non-pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto-regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation.

Superomedial Pedicle Vertical Scar Breast Reduction: Objective and Subjective Assessment of Breast Symmetry and Aesthetics.

BACKGROUND: The superomedial vertical scar breast reduction (SVBR) described by Hall-Findlay is gaining popularity among surgeons worldwide. The aim of this study was to evaluate its long-term aesthetic outcome, the extent of quality of life improvement and the factors that influence patient satisfaction and reviewers' evaluation of aesthetic/surgical outcome. METHODS: In this historical prospective study, we included women who underwent SVBR at least one year prior to enrollment and responded to a quality of life questionnaire. Their breasts were photographed, measured and evaluated by the plastic surgery staff. RESULTS: A total of 40 patients responded to the questionnaire, and the breasts of 31 of them were measured and photographed. All 31 patients had good breast symmetry according to objective breast measurements. There was a clear correlation between the patients' and the reviewers' scores of breast symmetry, scar appearance and breast shape (r = 0.4-0.65, r = 0.432-0.495 and r = 0.335-0.403, respectively). The factor that most influenced reviewers' and patients' satisfaction with the overall aesthetic outcome was the breast-to-body proportion. CONCLUSIONS: The proportions between the breast size and the patient's body habitus are pivotal to patient satisfaction and should be taken into consideration when planning a reduction mammaplasty. The SVBR technique for breast reduction provided good cosmetic outcome and symmetry over a long-term follow-up. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

DIEP Flap Breast Reconstruction Complication Rate in Previously Irradiated Internal Mammary Nodes.

BACKGROUND: Postmastectomy radiation therapy (PMRT) is a widely accepted treatment for locally advanced breast cancer. Some patients require additional boost radiation to the internal mammary nodes as the part of regional nodal irradiation (RNI). Delayed breast reconstruction with an autologous free flap using the internal mammary vessels for microvascular flap anastomosis is a common practice for these patients. The aim of our study was to evaluate the effect of RNI on autologous microvascular breast reconstruction. PATIENTS AND METHODS: A retrospective study was performed on 57 patients (69 flaps) undergoing deep inferior epigastric perforator (DIEP) flap for delayed breast reconstruction after radiation therapy. The study group included 37 patients (65%) who received PMRT and RNI to the internal mammary nodes. The control group included 20 patients (35%) who received PMRT alone. Early and delayed surgical complications, including wound complications, fat necrosis, and flap loss, were compared between the groups. RESULTS: The patient demographics in both groups were similar. Complication rate showed a higher trend in the study group for flap loss (8.3% versus 0%) and vascular anastomosis failure (5.6% versus 0%), but with no statistical difference (p = 0.54, 0.53, respectively). The control group showed a higher trend in fat necrosis (25% versus 8.3%, p = 0.11). CONCLUSIONS: DIEP flap breast reconstructions with internal mammary vessels anastomosis should be performed with cautious in patients who had received RNI to internal mammary nodes because of potential added risk for surgical complications.

Optimizing Patient Selection for Direct-to-Implant Immediate Breast Reconstruction Using Wise-Pattern Skin-Reducing Mastectomy in Large and Ptotic Breasts.

BACKGROUND: Immediate breast reconstruction in large and ptotic breasts is challenging, often requiring skin-reducing procedures. The Wise-pattern skin-reducing mastectomy (WPSRM) technique provides reliable one-stage implant coverage using the pectoralis muscle and a de-epithelialized inferiorly based dermal flap. However, de-vascularization may result in mastectomy skin flap necrosis. We aimed to critically evaluate and isolate patients at high risk of complications using this procedure. METHODS: We retrospectively reviewed consecutive patients undergoing WPSRM by the senior author from January 2008 to December 2011. Data collected included patient demographics, breast cancer staging, smoking, preoperative radiation, chemotherapy, BMI, mastectomy weight, implant size and type. We analyzed their effect on complications, revisions and failure rate. RESULTS: Fifty-nine WPSRMs were performed in 39 patients with a minimum of 12-month follow-up. Complications occurred in 43.75% patients and 34.88% breasts. Multivariate statistical analysis revealed that age (p = 0.093) and BMI (p = 0.631) were not significant risk factors as opposed to previously published data. Mastectomy weight was significantly associated with major complications requiring secondary surgery (odds ratio per 100 g of breast tissue was 1.18; 95% CI 1.01-1.39; p = 0.036) as 90.5% of our complications occurred in those patients with mastectomy weight exceeding 700 g. Complications were reduced twofold when selecting a Becker adjustable implant over a silicone one for these higher mastectomy weights. CONCLUSION: WPSRM was found to be associated with an increased complication rate in patients with high mastectomy weights and immediate reconstruction with silicone implants. We propose an algorithm offering better patient selection for this technique in immediate breast reconstruction. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Development of a fluorescence anisotropy-based assay for Dop, the first enzyme in the pupylation pathway.

The Pup-proteasome system (PPS) carries out regulated tagging and degradation of proteins in bacterial species belonging to the phyla Actinobacteria and Nitrospira. In the pathogen Mycobacterium tuberculosis, where this proteolytic pathway was initially discovered, PPS enzymes are essential for full virulence and persistence in the mammalian host. As such, PPS enzymes are potential targets for development of antituberculosis therapeutics. Such development often requires sensitive and robust assays for measurements of enzymatic activities and the effect of examined inhibitors. Here, we describe the development of an in vitro activity assay for Dop, the first enzyme in the PPS. Based on fluorescence anisotropy measurements, this assay is simple, sensitive, and compatible with a high-throughput format for screening purposes. We demonstrate how this assay can also be reliably and conveniently used for detailed kinetic measurements of Dop activity. As such, this assay is of value for basic research into Dop and the PPS. Finally, we show that the assay developed here primarily for the mycobacterial Dop can be readily employed with other Dop enzymes, using the same simple protocol.

The "Origami" Composite Free Fibula Flap for Complex Defects of the Mandible, Floor of the Mouth, and Tongue.

PURPOSE: The fibula flap serves as the workhorse for many mandibular defect types. For massive defects that include the mandible, floor of the mouth, and tongue, reconstruction might mandate a 2-flap approach. This report describes a systematic yet flexible design of a single composite osseocutaneous fibula flap for such cases. PATIENTS AND METHODS: From 2003 through 2011, 5 patients underwent surgery for T4 squamous cell carcinoma and adenoid cystic carcinoma. They underwent wide resection of the mandible and floor of the mouth with hemi- to subtotal glossectomy. A large skin paddle was designed on the leg using a customized template consisting of 3 components matching the alveolar, floor-of-mouth, and tongue segments. The round tongue component was folded using its thickness to provide 3-dimensional volume and form in place of the missing tongue. The other 2 components had a crescent shape and were adjusted intraoperatively to match the resected defect. RESULTS: All 5 flaps survived, with 1 case of flap loss that was successfully replaced. Delayed healing of the leg donor site was encountered in 4 patients. CONCLUSION: The proposed single 3-dimensionally folded flap design provides ample tissue to replace bony and soft tissue elements for major anterior or lateral segment defects. There was morbidity owing to donor-site complications but these were treatable, with recorded functional restoration.

Free fibula reconstruction of distal tibial defects after sarcoma surgery.

Distal tibial tumor ablation results in combined soft tissue and bone defect that involves the ankle joint. This area is unique and problematic because it combines low caliber limb size, relatively soft tissue deficiency, suboptimal bone and soft tissue healing ability, and the need to sustain increased mechanical loads. The management is difficult, controversial, and traditionally was treated by primary amputation. We present our experience with a limb sparing surgery using biological reconstruction.Between 2004 and 2007, 5 patients with malignant bone tumors of the distal tibia underwent tumor resection and reconstruction with free vascularized osteoseptocutaneous fibula flap. The average age was 33.2 years (range, 11-62 years). In all cases, a skin island was harvested and used for wound closure. Arthrodesis of the ankle joint was preformed in all patients. Double fixation system was used to provide stability and avoid flap rotation.All flaps survived. There were no major complications. One patient had partial loss of the skin paddle that was treated conservatively. Callous formation was documented after an average time of 4 months, partial weight bearing after an average time of 4 months, and full weight bearing after an average time of 11.5 months. All patients regained almost normal ambulation within a year.

Power-Assisted Liposuction Versus Tissue Resection for the Isolation of Adipose Tissue-Derived Mesenchymal Stem Cells: Phenotype, Senescence, and Multipotency at Advanced Passages.

BACKGROUND: Adipose tissue-derived mesenchymal stem cells (ASCs) can be isolated from subcutaneous fat harvested by tissue resection or liposuction. OBJECTIVES: The authors compared ASCs isolated by tissue resection or power-assisted liposuction (PAL) to determine whether either surgical procedure yielded ASCs with improved purity and competence that was preserved for several passages. METHODS: For this experimental study, ASCs were isolated from fat harvested by tissue resection or PAL from six patients who underwent abdominoplasty. ASCs were counted to determine cell yields, and viabilities were assessed with an amine-reactive dye and by fluorescence-activated cell sorting (FACS). Cell phenotypes were determined by immunostaining and FACS, and doubling times were calculated. Senescence ratios of the cells were detected by gene profiling and by assaying ß-galactosidase activity. Multipotency was evaluated by induced differentiation analyses. RESULTS: No significant differences were observed in cell numbers or viabilities of ASCs isolated following either surgical method of fat harvesting. Both populations of cultured ASCs expressed markers of mesenchymal stem cells and preserved this expression pattern through the third passage. PAL and tissue resection yielded ASCs with similar division rates, similar senescence ratios into the fourth passage, and similar capacities to differentiate into osteocytes or adipocytes. CONCLUSIONS: Fat harvested by PAL or tissue resection yielded uniform cultures of ASCs with high division rates, low senescence ratios, and multipotency preserved into passages 3 and 4. Because PAL is less invasive, it may be preferable for the isolation of ASCs.

Allosteric transitions direct protein tagging by PafA, the prokaryotic ubiquitin-like protein (Pup) ligase.

Protein degradation via prokaryotic ubiquitin-like protein (Pup) tagging is conserved in bacteria belonging to the phyla Actinobacteria and Nitrospira. The physiological role of this novel proteolytic pathway is not yet clear, although in Mycobacterium tuberculosis, the world's most threatening bacterial pathogen, Pup tagging is important for virulence. PafA, the Pup ligase, couples ATP hydrolysis with Pup conjugation to lysine side chains of protein substrates. PafA is the sole Pup ligase in M. tuberculosis and apparently, in other bacteria. Thus, whereas PafA is a key player in the Pup tagging (i.e. pupylation) system, control of its activity and interactions with target protein substrates remain poorly understood. In this study, we examined the mechanism of protein pupylation by PafA in Mycobacterium smegmatis, a model mycobacterial organism. We report that PafA is an allosteric enzyme that binds its target substrates cooperatively and find that PafA allostery is controlled by the binding of target protein substrates, yet is unaffected by Pup binding. Analysis of PafA pupylation using engineered substrates differing in the number of pupylation sites points to PafA acting as a dimer. These findings suggest that protein pupylation can be regulated at the level of PafA allostery.