Kip E. Guja, MD PhD

All Publications

• Molecular Imaging in Soft-tissue Sarcoma: Evolving Role of FDG PET. Seminars in nuclear medicine Guja, K. E., Ganjoo, K. N., Iagaru, A. 2024

  Abstract

  Soft tissue sarcomas are a rare and heterogenous group of tumors that account for 2% of all cancer-related deaths. Molecular imaging with FDG PET can offer valuable metabolic information to help inform clinical management of soft tissue sarcomas that is unique and complementary to conventional diagnostic imaging techniques. FDG PET imaging often correlates with tumor grade, can help guide biopsy, and frequently detects additional sites of disease compared to conventional imaging in patients being considered for definitive or salvage therapy. Traditional size-based evaluation of treatment response is often inadequate in soft tissue sarcoma and changes in metabolic activity can add significant value to interim and end of treatment imaging for high-grade sarcomas. FDG PET can be used for detection of recurrence or malignant transformation and thus play a vital role in surveillance. This article reviews the evolving role of FDG PET in initial diagnosis, staging, treatment response assessment, and restaging. Further studies on the use of FDG PET in soft sarcoma are needed, particularly for rare histopathologic subtypes.

  View details for DOI 10.1053/j.semnuclmed.2024.02.001

  View details for PubMedID 38433024

• Adverse Events in Targeted Radiopharmaceutical Therapy RadioGraphics Guja, K. E., Wu, J., Agordzo, H. L., Kwofie, J., Samaan, D., Nadel, H. R., Grady, E., Shah, J. 2024
• Molecular Imaging with PET/CT and PET/MRI in Pediatric Musculoskeletal Diseases Seminars in Nuclear Medicine Guja, K. E., Behr, G., Bedmutha, A., Kuhn, M., Nadel, H. R., Pandit-Taskar, N. 2024
• Overview and Recent Advances in 18F-FDG PET/CT for Evaluation of Pediatric Lymphoma Seminars in Nuclear Medicine Guja, K. E., Nadel, H., Iagaru, A. 2023; 53 (3): 400-412

  View details for DOI 10.1053/j.semnuclmed.2022.10.001

• Flip-flop: Two tracers are better than one in pediatric PET/MRI for neuroendocrine neoplasms Journal of Nuclear Medicine Guja, K. E., Nadel, H. R. 2023; 64 (1)
• Test Yourself: 7-month-old female with an enlarging left axillary mass. Skeletal radiology Guja, K. E., Hazard, F. K., Fadell, M. 2022

  View details for DOI 10.1007/s00256-022-04138-x

  View details for PubMedID 35913532

• PSMA theragnostics for metastatic castration resistant prostate cancer. Translational oncology Song, H., Guja, K. E., Iagaru, A. 2022; 22: 101438

  Abstract

  There has been tremendous growth in the development of theragnostics for personalized cancer diagnosis and treatment over the past two decades. In prostate cancer, the new generation of prostate specific membrane antigen (PSMA) small molecular inhibitor-based imaging agents achieve extraordinary tumor to background ratios and allow their therapeutic counterparts to deliver effective tumor doses while minimizing normal tissue toxicity. The PSMA targeted small molecule positron emission tomography (PET) agents 18F-DCFPyL (2-(3-{1-carboxy-5-((6-(18)F-fluoro-pyridine-3-carbonyl)-amino)-pentyl}-ureido)-pentanedioic acid) and Gallium-68 (68Ga)-PSMA-11 have been approved by the United States Food and Drug Administration (FDA) for newly diagnosed high risk prostate cancer patients and for patients with biochemical recurrence. More recently, the Phase III VISION trial showed that Lutetium-177 (177Lu)-PSMA-617 treatment increases progression-free survival and overall survival in patients with heavily pre-treated advanced PSMA-positive metastatic castration-resistant prostate cancer (mCRPC). Here, we review the PSMA targeted theragnostic pairs under clinical investigation for detection and treatment of metastatic prostate cancer.

  View details for DOI 10.1016/j.tranon.2022.101438

  View details for PubMedID 35659674

• Phantom study of SPECT/CT augmented reality for intraoperative localization of sentinel lymph nodes in head and neck melanoma. Oral oncology Nakamoto, R., Zhuo, J., Guja, K. E., Duan, H., Perkins, S. L., Leuze, C., Daniel, B. L., Franc, B. L. 1800; 125: 105702

  Abstract

  OBJECTIVE: To show that augmented reality (AR) visualization of single-photon emission computed tomography (SPECT)/computed tomography (CT) data in 3D can be used to accurately localize targets in the head and neck region.MATERIALS AND METHODS: Eight head and neck styrofoam phantoms were painted with a mixture of radioactive solution (Tc-99m) detectable with a handheld gamma probe and fluorescent ink visible only under ultraviolet (UV) light to create 10-20 simulated lymph nodes on their surface. After obtaining SPECT/CT images of these phantoms, virtual renderings of the nodes were generated from the SPECT/CT data and displayed using a commercially available AR headset. For each of three physician evaluators, the time required to localize lymph node targets was recorded (1) using the gamma probe alone and (2) using the gamma probe while wearing the AR headset. In addition, the surface localization accuracy when using the AR headset was evaluated by measuring the misalignment between the locations visually marked by the evaluators and the ground truth locations identified using UV stimulation of the ink at the site of the nodes.RESULTS: For all three evaluators, using the AR headset significantly reduced the time to detect targets (P=0.012, respectively) compared to using the gamma probe alone. The average misalignment between the location marked by the evaluators and the ground truth location was 8.6mm.CONCLUSION: AR visualization of SPECT/CT data in 3D allows for accurate localization of targets in the head and neck region, and may reduce the localization time of targets.

  View details for DOI 10.1016/j.oraloncology.2021.105702

  View details for PubMedID 34991004

• 64Cu-DOTATATE Uptake in a Pulmonary Hamartoma Clinical Nuclear Medicine Song, H., Guja, K. E., Yang, E. J., Guo, H. H. 2022; 48 (1): 58-60
• 18F-FDG PET/CT for Evaluation of Post-Transplant Lymphoproliferative Disorder (PTLD). Seminars in nuclear medicine Song, H., Guja, K. E., Iagaru, A. 2021

  Abstract

  Post-transplant lymphoproliferative disorders (PTLD) are a spectrum of heterogeneous lymphoproliferative conditions that are serious and possibly fatal complications after solid organ or allogenic hematopoietic stem cell transplantation. Most PTLD are attributed to Epstein-Barr virus reactivation in B-cells in the setting of immunosuppression after transplantation. Early diagnosis, accurate staging, and timely treatment are of vital importance to reduce morbidity and mortality. Given the often nonspecific clinical presentation and disease heterogeneity of PTLD, tissue biopsy and histopathological analysis are essential to establish diagnosis and most importantly, determine the subtype of PTLD, which guides treatment options. Advanced imaging modalities such as 18F-FDG PET/CT have played an increasingly important role and have shown high sensitivity and specificity in detection, staging, and assessing treatment response in multiple clinical studies over the last two decades. However, larger multicenter prospective validation is still needed to further establish the clinical utility of PET imaging in the management of PTLD. Significantly, new hybrid imaging modalities such as PET/MR may help reduce radiation exposure, which is especially important in pediatric transplant patients.

  View details for DOI 10.1053/j.semnuclmed.2020.12.009

  View details for PubMedID 33455722

• The Clinical Utility of 18F-Fluciclovine PET/CT in Biochemically Recurrent Prostate Cancer: an Academic Center Experience Post FDA Approval. Molecular imaging and biology Nakamoto, R. n., Harrison, C. n., Song, H. n., Guja, K. E., Hatami, N. n., Nguyen, J. n., Moradi, F. n., Franc, B. L., Aparici, C. M., Davidzon, G. n., Iagaru, A. n. 2021

  Abstract

  To evaluate the diagnostic performance and clinical utility of 18F-fluciclovine PET/CT in patients with biochemical recurrence (BCR) of prostate cancer (PC).18F-Fluciclovine scans of 165 consecutive men with BCR after primary definitive treatment with prostatectomy (n = 102) or radiotherapy (n = 63) were retrospectively evaluated. Seventy patients had concurrent imaging with at least one other conventional modality (CT (n = 31), MRI (n = 31), or bone scan (n = 26)). Findings from 18F-fluciclovine PET were compared with those from conventional imaging modalities. The positivity rate and impact of 18F-fluciclovine PET on patient management were recorded. In 33 patients who underwent at least one other PET imaging (18F-NaF PET/CT (n = 12), 68Ga-PSMA11 PET/CT (n = 5), 18F-DCFPyL PET/CT (n = 20), and 68Ga-RM2 PET/MRI (n = 5)), additional findings were evaluated.The overall positivity rate of 18F-fluciclovine PET was 67 %, which, as expected, increased with higher prostate-specific antigen (PSA) levels (ng/ml): 15 % (PSA < 0.5), 50 % (0.5 ≤ PSA < 1), 56 % (1 ≤ PSA < 2), 68 % (2 ≤ PSA < 5), and 94 % (PSA ≥ 5), respectively. One hundred and two patients (62 %) had changes in clinical management based on 18F-fluciclovine PET findings. Twelve of these patients (12 %) had lesion localization on 18F-fluciclovine PET, despite negative conventional imaging. Treatment plans of 14 patients with negative 18F-fluciclovine PET were changed based on additional PET imaging with a different radiopharmaceutical.18F-Fluciclovine PET/CT remains a useful diagnostic tool in the workup of patients with BCR PC, changing clinical management in 62 % of participants in our cohort.

  View details for DOI 10.1007/s11307-021-01583-3

  View details for PubMedID 33469884

• Prospective evaluation of F-18-DCFPyL PET/CT in biochemically recurrent prostate cancer: Analysis of lesion localization and distribution. Song, H., Duan, H., Harrison, C., Guja, K., Hatami, N., Franc, B., Moradi, F., Aparici, C., Davidzon, G., Srinivas, S., Iagaru, A. AMER SOC CLINICAL ONCOLOGY. 2020

  View details for Web of Science ID 000560368302399

• Peptide receptor radionuclide therapy (PRRT) for neuroendocrine tumors (NET): A two-year single institution experience Duan, H., Ninatti, G., Girod, B., Ferri, V., Guja, K., Song, H., Kunz, P., Fisher, G., Iagaru, A., Aparici, C. SOC NUCLEAR MEDICINE INC. 2020

  View details for Web of Science ID 000568290501374

• Fungal endocarditis resembling primary cardiac malignancy in a patient with B-cell ALL with culture confirmation. Radiology case reports Girod, B. J., Guja, K. E., Davidzon, G., Chan, F., Zucker, E., Franc, B. L., Moradi, F., Iagaru, A., Aparici, C. M. 2020; 15 (2): 117–19

  Abstract

  Fungal endocarditis is a rare subtype of infective endocarditis that often presents with nonspecific symptoms in patients with complex medical histories, making diagnosis challenging. Patients with a history of ALL may present with congestive heart failure, chemo-induced cardiomyopathy, acute coronary syndrome, cardiac lymphomatous metastasis, or infections. We present the case of a patient with a history of ALL who presented with acute coronary syndrome and imaging concerning for primary cardiac lymphoma, when in fact the patient ended up suffering from culture proven fungal endocarditis.

  View details for DOI 10.1016/j.radcr.2019.10.022

  View details for PubMedID 31768196

• An unusual presentation of recurrent T cell lymphoma: angiocentric pattern of cutaneous uptake on [18F]FDG PET/CT. European journal of nuclear medicine and molecular imaging Guja, K. E., Brown, R. n., Girod, B. n., Song, H. n., Harrison, C. n., Franc, B. L., Moradi, F. n., Davidzon, G. n., Iagaru, A. n., Aparici, C. M. 2020

  View details for DOI 10.1007/s00259-020-05026-z

  View details for PubMedID 32918110

• Prospective Evaluation in an Academic Center of 18F-DCFPyL PET/CT in Biochemically Recurrent Prostate Cancer: A Focus on Localizing Disease and Changes in Management. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Song, H., Harrison, C., Duan, H., Guja, K., Hatami, N., Franc, B., Moradi, F., Mari Aparici, C., Davidzon, G., Iagaru, A. 2019

  Abstract

  18F-DCFPyL is a promising PET radiopharmaceutical targeting prostate specific membrane antigen (PSMA). We present our experience in this single academic center prospective study evaluating the positivity rate of 18F-DCFPyL PET/CT in patients with biochemical recurrence (BCR) of prostate cancer (PC). Methods: We prospectively enrolled 72 men (52-91 years old, mean±SD: 71.5±7.2) with BCR after primary definitive treatment with prostatectomy (n = 42) or radiotherapy (n = 30). The presence of lesions compatible with PC was evaluated by two independent readers. Fifty-nine patients had concurrent scans with at least one other conventional scan: bone scan (24), CT (21), MR (20), 18F-Fluciclovine PET/CT (18) and/or 18F-NaF PET (14). Findings from 18F-DCFPyL PET/CT were compared with those from other modalities. Impact on patient management based on 18F-DCFPyL PET/CT was recorded from clinical chart review. Results: 18F-DCFPyL PET/CT had an overall positivity rate of 85%, which increased with higher prostate specific antigen (PSA) levels (ng/mL): 50% (PSA<0.5), 69% (0.5≤PSA<1), 100% (1≤PSA<2), 91% (2≤PSA<5) and 96% (PSA≥5), respectively. 18F-DCFPyL PET detected more lesions than conventional imaging. For anatomic imaging, 20/41 (49%) CT/MRI had congruent findings with 18F-DCFPyL, while 18F-DCFPyL PET was positive in 17/41 (41%) cases with negative CT/MRI. For bone imaging, 26/38 (68%) bone scan/18F-NaF PET were congruent with 18F-DCFPyL PET, while 18F-DCFPyL PET localized bone lesions in 8/38 (21%) patients with negative bone scan/18F-NaF PET. In 8/18 (44%) patients, 18F-Fluciclovine PET had located the same lesions as the 18F-DCFPyL PET, while 5/18 (28%) patients with negative 18F-Fluciclovine had positive 18F-DCFPyL PET findings and 1/18 (6%) patient with negative 18F-DCFPyL had uptake in the prostate bed on 18F-Fluciclovine PET. In the remaining 4/18 (22%) patients, 18F-DCFPyL and 18F-Fluciclovine scans showed different lesions. Lastly, 43/72 (60%) patients had treatment changes after 18F-DCFPyL PET and, most noticeably, 17 of these patients (24% total) had lesion localization only on 18F-DCFPyL PET, despite negative conventional imaging. Conclusion: 18F-DCFPyL PET/CT is a promising diagnostic tool in the work-up of biochemically recurrent prostate cancer given the high positivity rate as compared to FDA-approved currently available imaging modalities and its impact on clinical management in 60% of patients.

  View details for DOI 10.2967/jnumed.119.231654

  View details for PubMedID 31628216

• HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression (vol 2, 16045, 2016) CELL DISCOVERY Guha, M., Srinivasan, S., Guja, K., Mejia, E., Garcia-Diaz, M., Johnson, F., Ruthel, G., Kaufman, B. A., Rappaport, E. F., Glineburg, M., Fang, J., Klein-Szanto, A. J., Nakagawa, H., Basha, J., Kundu, T., Avadhani, N. G. 2019; 5: 28

  Abstract

  [This corrects the article DOI: 10.1038/celldisc.2016.45.].

  View details for DOI 10.1038/s41421-019-0097-7

  View details for Web of Science ID 000468984600001

  View details for PubMedID 31098295

  View details for PubMedCentralID PMC6517377

• Prospective Evaluation of F-18-DCFPyL PET/CT and Conventional Imaging in Patients with Biochemically Recurrent Prostate Cancer Song, H., Harrison, C., Guja, K., Franc, B., Moradi, F., Davidzon, G., Aparici, C., Iagaru, A. SOC NUCLEAR MEDICINE INC. 2019

  View details for Web of Science ID 000473116801016

• Prospective evaluation of F-18- DCFPyL in Patients with Biochemically Recurrent Prostate Cancer: Positivity Rate and Correlation with PSA levels Harrison, C., Song, H., Franc, B. L., Guja, K., Moradi, F., Davidzon, G., Aparici, C., Iagaru, A. SOC NUCLEAR MEDICINE INC. 2019

  View details for Web of Science ID 000473116801610

• Quantification of uptake in Ga-68-DOTATATE PET: Correlation between standardized uptake values and patient factors Moradi, F., Guja, K., Aparici, C., Iagaru, A. SOC NUCLEAR MEDICINE INC. 2019

  View details for Web of Science ID 000473116800434

• hnRNPA2 mediated acetylation reduces telomere length in response to mitochondrial dysfunction. PloS one Guha, M., Srinivasan, S., Johnson, F. B., Ruthel, G., Guja, K., Garcia-Diaz, M., Kaufman, B. A., Glineburg, M. R., Fang, J., Nakagawa, H., Basha, J., Kundu, T., Avadhani, N. G. 2018; 13 (11): e0206897

  Abstract

  Telomeres protect against chromosomal damage. Accelerated telomere loss has been associated with premature aging syndromes such as Werner's syndrome and Dyskeratosis Congenita, while, progressive telomere loss activates a DNA damage response leading to chromosomal instability, typically observed in cancer cells and senescent cells. Therefore, identifying mechanisms of telomere length maintenance is critical for understanding human pathologies. In this paper we demonstrate that mitochondrial dysfunction plays a causal role in telomere shortening. Furthermore, hnRNPA2, a mitochondrial stress responsive lysine acetyltransferase (KAT) acetylates telomere histone H4at lysine 8 of (H4K8) and this acetylation is associated with telomere attrition. Cells containing dysfunctional mitochondria have higher telomere H4K8 acetylation and shorter telomeres independent of cell proliferation rates. Ectopic expression of KAT mutant hnRNPA2 rescued telomere length possibly due to impaired H4K8 acetylation coupled with inability to activate telomerase expression. The phenotypic outcome of telomere shortening in immortalized cells included chromosomal instability (end-fusions) and telomerase activation, typical of an oncogenic transformation; while in non-telomerase expressing fibroblasts, mitochondrial dysfunction induced-telomere attrition resulted in senescence. Our findings provide a mechanistic association between dysfunctional mitochondria and telomere loss and therefore describe a novel epigenetic signal for telomere length maintenance.

  View details for DOI 10.1371/journal.pone.0206897

  View details for PubMedID 30427907

  View details for PubMedCentralID PMC6241121

• Response to Nazarian et al regarding: "Cost-effectiveness of magnetic resonance imaging versus ultrasound for the detection of symptomatic full-thickness supraspinatus tendon tears" JOURNAL OF SHOULDER AND ELBOW SURGERY Gyftopoulos, S., Guja, K. E., Subhas, N., Virk, M. S., Gold, H. T. 2018; 27 (10): E320-E321

  View details for DOI 10.1016/j.jse.2018.03.034

  View details for Web of Science ID 000444876000007

  View details for PubMedID 30093231

• Cost-effectiveness of magnetic resonance imaging versus ultrasound for the detection of symptomatic full-thickness supraspinatus tendon tears. Journal of shoulder and elbow surgery Gyftopoulos, S., Guja, K. E., Subhas, N., Virk, M. S., Gold, H. T. 2017; 26 (12): 2067-2077

  Abstract

  The purpose of this study was to determine the value of magnetic resonance imaging (MRI) and ultrasound-based imaging strategies in the evaluation of a hypothetical population with a symptomatic full-thickness supraspinatus tendon (FTST) tear using formal cost-effectiveness analysis.A decision analytic model from the health care system perspective for 60-year-old patients with symptoms secondary to a suspected FTST tear was used to evaluate the incremental cost-effectiveness of 3 imaging strategies during a 2-year time horizon: MRI, ultrasound, and ultrasound followed by MRI. Comprehensive literature search and expert opinion provided data on cost, probability, and quality of life estimates. The primary effectiveness outcome was quality-adjusted life-years (QALYs) through 2 years, with a willingness-to-pay threshold set to $100,000/QALY gained (2016 U.S. dollars). Costs and health benefits were discounted at 3%.Ultrasound was the least costly strategy ($1385). MRI was the most effective (1.332 QALYs). Ultrasound was the most cost-effective strategy but was not dominant. The incremental cost-effectiveness ratio for MRI was $22,756/QALY gained, below the willingness-to-pay threshold. Two-way sensitivity analysis demonstrated that MRI was favored over the other imaging strategies over a wide range of reasonable costs. In probabilistic sensitivity analysis, MRI was the preferred imaging strategy in 78% of the simulations.MRI and ultrasound represent cost-effective imaging options for evaluation of the patient thought to have a symptomatic FTST tear. The results indicate that MRI is the preferred strategy based on cost-effectiveness criteria, although the decision between MRI and ultrasound for an imaging center is likely to be dependent on additional factors, such as available resources and workflow.

  View details for DOI 10.1016/j.jse.2017.07.012

  View details for PubMedID 28893546

• Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation. Proceedings of the National Academy of Sciences of the United States of America Airola, M. V., Shanbhogue, P., Shamseddine, A. A., Guja, K. E., Senkal, C. E., Maini, R., Bartke, N., Wu, B. X., Obeid, L. M., Garcia-Diaz, M., Hannun, Y. A. 2017; 114 (28): E5549-E5558

  Abstract

  Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I-type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the "DK switch." Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.

  View details for DOI 10.1073/pnas.1705134114

  View details for PubMedID 28652336

  View details for PubMedCentralID PMC5514751

• HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression. Cell discovery Guha, M., Srinivasan, S., Guja, K., Mejia, E., Garcia-Diaz, M., Johnson, F. B., Ruthel, G., Kaufman, B. A., Rappaport, E. F., Glineburg, M. R., Fang, J. K., Klein-Szanto, A. J., Klein Szanto, A., Nakagawa, H., Basha, J., Kundu, T., Avadhani, N. G. 2016; 2: 16045

  Abstract

  Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies.

  View details for DOI 10.1038/celldisc.2016.45

  View details for PubMedID 27990297

  View details for PubMedCentralID PMC5148442

• Structural and Biochemical Basis for Intracellular Kinase Inhibition by Src-specific Peptidic Macrocycles. Cell chemical biology Aleem, S., Georghiou, G., Kleiner, R. E., Guja, K., Craddock, B. P., Lyczek, A., Chan, A. I., Garcia-Diaz, M., Miller, W. T., Liu, D. R., Seeliger, M. A. 2016; 23 (9): 1103-1112

  Abstract

  Protein kinases are attractive therapeutic targets because their dysregulation underlies many diseases, including cancer. The high conservation of the kinase domain and the evolution of drug resistance, however, pose major challenges to the development of specific kinase inhibitors. We recently discovered selective Src kinase inhibitors from a DNA-templated macrocycle library. Here, we reveal the structural basis for how these inhibitors retain activity against a disease-relevant, drug-resistant kinase mutant, while maintaining Src specificity. We find that these macrocycles display a degree of modularity: two of their three variable groups interact with sites on the kinase that confer selectivity, while the third group interacts with the universally conserved catalytic lysine and thereby retains the ability to inhibit the "gatekeeper" kinase mutant. We also show that these macrocycles inhibit migration of MDA-MB-231 breast tumor cells. Our findings establish intracellular kinase inhibition by peptidic macrocycles, and inform the development of potent and specific kinase inhibitors.

  View details for DOI 10.1016/j.chembiol.2016.07.017

  View details for PubMedID 27593110

  View details for PubMedCentralID PMC5096457

• A fidelity mechanism in DNA polymerase lambda promotes error-free bypass of 8-oxo-dG. The EMBO journal Burak, M. J., Guja, K. E., Hambardjieva, E., Derkunt, B., Garcia-Diaz, M. 2016; 35 (18): 2045-59

  Abstract

  8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxo-dG) has high mutagenic potential as it is prone to mispair with deoxyadenine (dA). In order to maintain genomic integrity, post-replicative 8-oxo-dG:dA mispairs are removed through DNA polymerase lambda (Pol λ)-dependent MUTYH-initiated base excision repair (BER). Here, we describe seven novel crystal structures and kinetic data that fully characterize 8-oxo-dG bypass by Pol λ. We demonstrate that Pol λ has a flexible active site that can tolerate 8-oxo-dG in either the anti- or syn-conformation. Importantly, we show that discrimination against the pro-mutagenic syn-conformation occurs at the extension step and identify the residue responsible for this selectivity. This residue acts as a kinetic switch, shunting repair toward long-patch BER upon correct dCMP incorporation, thus enhancing repair efficiency. Moreover, this switch also provides a potential mechanism to increase repair fidelity of MUTYH-initiated BER.

  View details for DOI 10.15252/embj.201694332

  View details for PubMedID 27481934

  View details for PubMedCentralID PMC5282837

• Reviewing the Reviewers: The Timeliness of Peer Review in Radiology Journals Guja, K. E., Janardhanan, A., Stavro, J., Castillo, M., Schweitzer, M. E. Radiological Society of North America. 2016
• Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda. Nucleic acids research Burak, M. J., Guja, K. E., Garcia-Diaz, M. 2015; 43 (16): 8089-99

  Abstract

  8-Oxo-7,8,-dihydro-2'-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala(510) and Asn(513), play differential roles in dNTP selectivity. Specifically, Ala(510) and Asn(513) facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases.

  View details for DOI 10.1093/nar/gkv760

  View details for PubMedID 26220180

  View details for PubMedCentralID PMC4652769

• Completing the specificity swap: Single-stranded DNA recognition by F and R100 TraI relaxase domains. Plasmid Guja, K. E., Schildbach, J. F. 2015; 80: 1-7

  Abstract

  During conjugative plasmid transfer, one plasmid strand is cleaved and transported to the recipient bacterium. For F and related plasmids, TraI contains the relaxase or nickase activity that cleaves the plasmid DNA strand. F TraI36, the F TraI relaxase domain, binds a single-stranded origin of transfer (oriT) DNA sequence with high affinity and sequence specificity. The TraI36 domain from plasmid R100 shares 91% amino acid sequence identity with F TraI36, but its oriT DNA binding site differs by two of eleven bases. Both proteins readily distinguish between F and R100 binding sites. In earlier work, two amino acid substitutions in the DNA binding cleft were shown to be sufficient to change the R100 TraI36 DNA-binding specificity to that of F TraI36. In contrast, three substitutions could make F TraI36 more "R100-like", but failed to completely alter the specificity. Here we identify one additional amino acid substitution that completes the specificity swap from F to R100. To our surprise, adding further substitutions from R100 to the F background were detrimental to binding instead of being neutral, indicating that their effects were influenced by their structural context. These results underscore the complex and subtle nature of DNA recognition by relaxases and have implications for the evolution of relaxase binding sites and oriT sequences.

  View details for DOI 10.1016/j.plasmid.2015.03.006

  View details for PubMedID 25841886

• Unraveling Cholesterol Catabolism in Mycobacterium tuberculosis: ChsE4-ChsE5 α2β2 Acyl-CoA Dehydrogenase Initiates β-Oxidation of 3-Oxo-cholest-4-en-26-oyl CoA. ACS infectious diseases Yang, M., Lu, R., Guja, K. E., Wipperman, M. F., St Clair, J. R., Bonds, A. C., Garcia-Diaz, M., Sampson, N. S. 2015; 1 (2): 110-125

  Abstract

  The metabolism of host cholesterol by Mycobacterium tuberculosis (Mtb) is an important factor for both its virulence and pathogenesis, although how and why cholesterol metabolism is required is not fully understood. Mtb uses a unique set of catabolic enzymes that are homologous to those required for classical β-oxidation of fatty acids but are specific for steroid-derived substrates. Here, we identify and assign the substrate specificities of two of these enzymes, ChsE4-ChsE5 (Rv3504-Rv3505) and ChsE3 (Rv3573c), that carry out cholesterol side chain oxidation in Mtb. Steady-state assays demonstrate that ChsE4-ChsE5 preferentially catalyzes the oxidation of 3-oxo-cholest-4-en-26-oyl CoA in the first cycle of cholesterol side chain β-oxidation that ultimately yields propionyl-CoA, whereas ChsE3 specifically catalyzes the oxidation of 3-oxo-chol-4-en-24-oyl CoA in the second cycle of β-oxidation that generates acetyl-CoA. However, ChsE4-ChsE5 can catalyze the oxidation of 3-oxo-chol-4-en-24-oyl CoA as well as 3-oxo-4-pregnene-20-carboxyl-CoA. The functional redundancy of ChsE4-ChsE5 explains the in vivo phenotype of the igr knockout strain of Mycobacterium tuberculosis; the loss of ChsE1-ChsE2 can be compensated for by ChsE4-ChsE5 during the chronic phase of infection. The X-ray crystallographic structure of ChsE4-ChsE5 was determined to a resolution of 2.0 Å and represents the first high-resolution structure of a heterotetrameric acyl-CoA dehydrogenase (ACAD). Unlike typical homotetrameric ACADs that bind four flavin adenine dinucleotide (FAD) cofactors, ChsE4-ChsE5 binds one FAD at each dimer interface, resulting in only two substrate-binding sites rather than the classical four active sites. A comparison of the ChsE4-ChsE5 substrate-binding site to those of known mammalian ACADs reveals an enlarged binding cavity that accommodates steroid substrates and highlights novel prospects for designing inhibitors against the committed β-oxidation step in the first cycle of cholesterol side chain degradation by Mtb.

  View details for DOI 10.1021/id500033m

  View details for PubMedID 26161441

  View details for PubMedCentralID PMC4489319

• A distinct MaoC-like enoyl-CoA hydratase architecture mediates cholesterol catabolism in Mycobacterium tuberculosis. ACS chemical biology Yang, M., Guja, K. E., Thomas, S. T., Garcia-Diaz, M., Sampson, N. S. 2014; 9 (11): 2632-45

  Abstract

  The Mycobacterium tuberculosis (Mtb) igr operon plays an essential role in Mtb cholesterol metabolism, which is critical for pathogenesis during the latent stage of Mtb infection. Here we report the first structure of a heterotetrameric MaoC-like enoyl-CoA hydratase, ChsH1-ChsH2, which is encoded by two adjacent genes from the igr operon. We demonstrate that ChsH1-ChsH2 catalyzes the hydration of a steroid enoyl-CoA, 3-oxo-4,17-pregnadiene-20-carboxyl-CoA, in the modified β-oxidation pathway for cholesterol side chain degradation. The ligand-bound and apoenzyme structures of ChsH1-ChsH2(N) reveal an unusual, modified hot-dog fold with a severely truncated central α-helix that creates an expanded binding site to accommodate the bulkier steroid ring system. The structures show quaternary structure shifts that accommodate the four rings of the steroid substrate and offer an explanation for why the unusual heterotetrameric assembly is utilized for hydration of this steroid. The unique αβ heterodimer architecture utilized by ChsH1-ChsH2 to bind its distinctive substrate highlights an opportunity for the development of new antimycobacterial drugs that target a pathway specific to Mtb.

  View details for DOI 10.1021/cb500232h

  View details for PubMedID 25203216

  View details for PubMedCentralID PMC4245171

• Organization of the human mitochondrial transcription initiation complex. Nucleic acids research Yakubovskaya, E., Guja, K. E., Eng, E. T., Choi, W. S., Mejia, E., Beglov, D., Lukin, M., Kozakov, D., Garcia-Diaz, M. 2014; 42 (6): 4100-12

  Abstract

  Initiation of transcription in human mitochondria involves two factors, TFAM and TFB2M, in addition to the mitochondrial RNA polymerase, POLRMT. We have investigated the organization of the human mitochondrial transcription initiation complex on the light-strand promoter (LSP) through solution X-ray scattering, electron microscopy (EM) and biochemical studies. Our EM results demonstrate a compact organization of the initiation complex, suggesting that protein-protein interactions might help mediate initiation. We demonstrate that, in the absence of DNA, only POLRMT and TFAM form a stable interaction, albeit one with low affinity. This is consistent with the expected transient nature of the interactions necessary for initiation and implies that the promoter DNA acts as a scaffold that enables formation of the full initiation complex. Docking of known crystal structures into our EM maps results in a model for transcriptional initiation that strongly correlates with new and existing biochemical observations. Our results reveal the organization of TFAM, POLRMT and TFB2M around the LSP and represent the first structural characterization of the entire mitochondrial transcriptional initiation complex.

  View details for DOI 10.1093/nar/gkt1360

  View details for PubMedID 24413562

  View details for PubMedCentralID PMC3973321

• Non-stop mRNA decay: a special attribute of trans-translation mediated ribosome rescue. Frontiers in microbiology Venkataraman, K., Guja, K. E., Garcia-Diaz, M., Karzai, A. W. 2014; 5: 93

  Abstract

  Decoding of aberrant mRNAs leads to unproductive ribosome stalling and sequestration of components of the translation machinery. Bacteria have evolved three seemingly independent pathways to resolve stalled translation complexes. The trans-translation process, orchestrated by the hybrid transfer-messenger RNA (tmRNA) and its essential protein co-factor, small protein B (SmpB), is the principal translation quality control system for rescuing unproductively stalled ribosomes. Two specialized alternative rescue pathways, coordinated by ArfA and ArfB, have been recently discovered. The SmpB-tmRNA mediated trans-translation pathway, in addition to re-mobilizing stalled translation complexes, co-translationally appends a degradation tag to the associated nascent polypeptides, marking them for proteolysis by various cellular proteases. Another unique feature of trans-translation, not shared by the alternative rescue pathways, is the facility to recruit ribonuclease R (RNase R) for targeted degradation of non-stop mRNAs, thus preventing further futile cycles of translation. The distinct C-terminal lysine-rich (K-rich) domain of RNase R is essential for its recruitment to stalled ribosomes. To gain new insights into the structure and function of RNase R, we investigated its global architecture, the spatial arrangement of its distinct domains, and the identities of key functional residues in its unique K-rich domain. Small-angle X-ray scattering models of RNase R reveal a tri-lobed structure with flexible N- and C-terminal domains, and suggest intimate contacts between the K-rich domain and the catalytic core of the enzyme. Alanine-scanning mutagenesis of the K-rich domain, in the region spanning residues 735 and 750, has uncovered the precise amino acid determinants required for the productive engagement of RNase R on tmRNA-rescued ribosomes. Theses analyses demonstrate that alanine substitution of conserved residues E740 and K741result in profound defects, not only in the recruitment of RNase R to rescued ribosomes but also in the targeted decay of non-stop mRNAs. Additionally, an RNase R variant with alanine substitution at residues K749 and K750 exhibits extensive defects in ribosome enrichment and non-stop mRNA decay. In contrast, alanine substitution of additional conserved residues in this region has no effect on the known functions of RNase R. In vitro RNA degradation assays demonstrate that the consequential substitutions (RNase R(E740A/K741A) and RNase R(K749A/K750A)) do not affect the ability of the enzyme to degrade structured RNAs, indicating that the observed defect is specific to the trans-translation related activities of RNase R. Taken together, these findings shed new light on the global architecture of RNase R and provide new details of how this versatile RNase effectuates non-stop mRNA decay on tmRNA-rescued ribosomes.

  View details for DOI 10.3389/fmicb.2014.00093

  View details for PubMedID 24653719

  View details for PubMedCentralID PMC3949413

• A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site. PloS one Jacewicz, A., Trzemecka, A., Guja, K. E., Plochocka, D., Yakubovskaya, E., Bebenek, A., Garcia-Diaz, M. 2013; 8 (10): e76700

  Abstract

  Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A) mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.

  View details for DOI 10.1371/journal.pone.0076700

  View details for PubMedID 24116139

  View details for PubMedCentralID PMC3792054

• Structural basis for S-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor B1. Nucleic acids research Guja, K. E., Venkataraman, K., Yakubovskaya, E., Shi, H., Mejia, E., Hambardjieva, E., Karzai, A. W., Garcia-Diaz, M. 2013; 41 (16): 7947-59

  Abstract

  Eukaryotic transcription factor B (TFB) proteins are homologous to KsgA/Dim1 ribosomal RNA (rRNA) methyltransferases. The mammalian TFB1, mitochondrial (TFB1M) factor is an essential protein necessary for mitochondrial gene expression. TFB1M mediates an rRNA modification in the small ribosomal subunit and thus plays a role analogous to KsgA/Dim1 proteins. This modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and diabetes. Here, we present the first structural characterization of the mammalian TFB1 factor. We have solved two X-ray crystallographic structures of TFB1M with (2.1 Å) and without (2.0 Å) its cofactor S-adenosyl-L-methionine. These structures reveal that TFB1M shares a conserved methyltransferase core with other KsgA/Dim1 methyltransferases and shed light on the structural basis of S-adenosyl-L-methionine binding and methyltransferase activity. Together with mutagenesis studies, these data suggest a model for substrate binding and provide insight into the mechanism of methyl transfer, clarifying the role of this factor in an essential process for mitochondrial function.

  View details for DOI 10.1093/nar/gkt547

  View details for PubMedID 23804760

  View details for PubMedCentralID PMC3763538

• Structure of the essential MTERF4:NSUN4 protein complex reveals how an MTERF protein collaborates to facilitate rRNA modification. Structure (London, England : 1993) Yakubovskaya, E., Guja, K. E., Mejia, E., Castano, S., Hambardjieva, E., Choi, W. S., Garcia-Diaz, M. 2012; 20 (11): 1940-7

  Abstract

  MTERF4 is the first MTERF family member shown to bind RNA and plays an essential role as a regulator of ribosomal biogenesis in mammalian mitochondria. It forms a complex with the rRNA methyltransferase NSUN4 and recruits it to the large ribosomal subunit. In this article, we characterize the interaction between both proteins, demonstrate that MTERF4 strongly stimulates the specificity of NSUN4 during in vitro methylation experiments, and present the 2.0 Å resolution crystal structure of the MTERF4:NSUN4 protein complex, lacking 48 residues of the MTERF4 C-terminal acidic tail, bound to S-adenosyl-L-methionine, thus revealing the nature of the interaction between both proteins and the structural conservation of the most divergent of the human MTERF family members. Moreover, the structure suggests a model for RNA binding by the MTERF4:NSUN4 complex, providing insight into the mechanism by which an MTERF family member facilitates rRNA methylation.

  View details for DOI 10.1016/j.str.2012.08.027

  View details for PubMedID 23022348

  View details for PubMedCentralID PMC3496059

• Hitting the brakes: termination of mitochondrial transcription. Biochimica et biophysica acta Guja, K. E., Garcia-Diaz, M. 2011; 1819 (9-10): 939-47

  Abstract

  Deficiencies in mitochondrial protein production are associated with human disease and aging. Given the central role of transcription in gene expression, recent years have seen a renewed interest in understanding the molecular mechanisms controlling this process. In this review, we have focused on the mostly uncharacterized process of transcriptional termination. We review how several recent breakthroughs have provided insight into our understanding of the termination mechanism, the protein factors that mediate termination, and the functional relevance of different termination events. Furthermore, the identification of termination defects resulting from a number of mtDNA mutations has led to the suggestion that this could be a common mechanism influencing pathogenesis in a number of mitochondrial diseases, highlighting the importance of understanding the processes that regulate transcription in human mitochondria. We discuss how these recent findings set the stage for future studies on this important regulatory mechanism. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

  View details for DOI 10.1016/j.bbagrm.2011.11.004

  View details for PubMedID 22137970

  View details for PubMedCentralID PMC3408806

• An intrastrand three-DNA-base interaction is a key specificity determinant of F transfer initiation and of F TraI relaxase DNA recognition and cleavage. Nucleic acids research Hekman, K., Guja, K., Larkin, C., Schildbach, J. F. 2008; 36 (14): 4565-72

  Abstract

  Bacterial conjugation, transfer of a single conjugative plasmid strand between bacteria, diversifies prokaryotic genomes and disseminates antibiotic resistance genes. As a prerequisite for transfer, plasmid-encoded relaxases bind to and cleave the transferred plasmid strand with sequence specificity. The crystal structure of the F TraI relaxase domain with bound single-stranded DNA suggests binding specificity is partly determined by an intrastrand three-way base-pairing interaction. We showed previously that single substitutions for the three interacting bases could significantly reduce binding. Here we examine the effect of single and double base substitutions at these positions on plasmid mobilization. Many substitutions reduce transfer, although the detrimental effects of some substitutions can be partially overcome by substitutions at a second site. We measured the affinity of the F TraI relaxase domain for several DNA sequence variants. While reduced transfer generally correlates with reduced binding affinity, some oriT variants transfer with an efficiency different than expected from their binding affinities, indicating ssDNA binding and cleavage do not correlate absolutely. Oligonucleotide cleavage assay results suggest the essential function of the three-base interaction may be to position the scissile phosphate for cleavage, rather than to directly contribute to binding affinity.

  View details for DOI 10.1093/nar/gkn422

  View details for PubMedID 18611948

  View details for PubMedCentralID PMC2504302

• Using fluorophore-labeled oligonucleotides to measure affinities of protein-DNA interactions. Methods in enzymology Anderson, B. J., Larkin, C., Guja, K., Schildbach, J. F. 2008; 450: 253-72

  Abstract

  Changes in fluorescence emission intensity and anisotropy can reflect changes in the environment and molecular motion of a fluorophore. Researchers can capitalize on these characteristics to assess the affinity and specificity of DNA-binding proteins using fluorophore-labeled oligonucleotides. While there are many advantages to measuring binding using fluorescent oligonucleotides, there are also some distinct disadvantages. Here we describe some of the relevant issues for the novice, illustrating key points using data collected with a variety of labeled oligonucleotides and the relaxase domain of F plasmid TraI. Topics include selection of a fluorophore, experimental design using a fluorometer equipped with an automatic titrating unit, and analysis of direct binding and competition assays.

  View details for DOI 10.1016/S0076-6879(08)03412-5

  View details for PubMedID 19152864

  View details for PubMedCentralID PMC3051356